U.S. patent application number 17/463156 was filed with the patent office on 2022-05-12 for methods for increasing efficacy of folr1 cancer therapy.
This patent application is currently assigned to ImmunoGen, Inc.. The applicant listed for this patent is ImmunoGen, Inc.. Invention is credited to Christina N. CARRIGAN, Sharron LADD, Gillian PAYNE, Kathleen R. WHITEMAN.
Application Number | 20220143208 17/463156 |
Document ID | / |
Family ID | 1000006096181 |
Filed Date | 2022-05-12 |
United States Patent
Application |
20220143208 |
Kind Code |
A1 |
CARRIGAN; Christina N. ; et
al. |
May 12, 2022 |
METHODS FOR INCREASING EFFICACY OF FOLR1 CANCER THERAPY
Abstract
Methods to improve the success of cancer therapies that target
the human folate receptor 1 are provided. Kits comprising reagent
useful in the methods are further provided.
Inventors: |
CARRIGAN; Christina N.; (San
Francisco, CA) ; WHITEMAN; Kathleen R.; (Wilmington,
MA) ; PAYNE; Gillian; (Waban, MA) ; LADD;
Sharron; (Gregory, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ImmunoGen, Inc. |
Waltham |
MA |
US |
|
|
Assignee: |
ImmunoGen, Inc.
Waltham
MA
|
Family ID: |
1000006096181 |
Appl. No.: |
17/463156 |
Filed: |
August 31, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15807831 |
Nov 9, 2017 |
11135305 |
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17463156 |
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14245797 |
Apr 4, 2014 |
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15807831 |
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13435857 |
Mar 30, 2012 |
8709432 |
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14245797 |
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61471007 |
Apr 1, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6849 20170801;
A61K 39/395 20130101; A61K 47/6851 20170801; C07K 16/28 20130101;
A61K 2039/505 20130101; G01N 33/577 20130101; G01N 33/57492
20130101; G01N 2800/52 20130101; A61K 47/6809 20170801; A61K
47/6869 20170801 |
International
Class: |
A61K 47/68 20060101
A61K047/68; C07K 16/28 20060101 C07K016/28; G01N 33/574 20060101
G01N033/574 |
Claims
1.-19. (canceled)
20. An article of manufacture comprising an anti-FOLR1 antibody or
an anti-FOLR1 immunoconjugate; a container; and a package insert or
label indicating that the antibody or immunoconjugate can be used
to treat a cancer characterized by the expression of FOLR1 at a
level of 2, 3, or 3+ measured by IHC.
21.-96. (canceled)
97. A method for treating ovarian cancer comprising administering a
therapeutically effective dose of an anti-Folate Receptor 1 (FOLR1)
immunoconjugate to a human subject having ovarian cancer; wherein a
FOLR1 staining intensity score of 2 or greater has been detected in
greater than 25% of the cells in a tumor sample from the human
subject using an immunohistochemistry (IHC) detection method that
distinguishes between staining intensity and staining
uniformity.
98. The method of claim 97, wherein the anti-FOLR1 immunoconjugate
has the formula (A)-(L)-(C), wherein: (A) comprises an antibody or
antigen binding fragment thereof comprising: (a) a heavy chain CDR1
comprising the amino acid sequence GYFMN (SEQ ID NO:6); a heavy
chain CDR2 comprising the amino acid sequence RIHPYDGDTFYNQKFQG
(SEQ ID NO:7); and a heavy chain CDR3 comprising the amino acid
sequence YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1
comprising the amino acid sequence KASQSVSFAGTSLMH (SEQ ID NO:9); a
light chain CDR2 comprising the amino acid sequence RASNLEA (SEQ ID
NO:10); and a light chain CDR3 comprising the amino acid sequence
QQSREYPYT (SEQ ID NO:11), (L) comprises the linker N-succinimidyl
4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), and (C)
comprises the cytotoxic agent
N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine
(DM4), and wherein the linker (L) links (A) to (C).
99. The method of claim 99, wherein the antibody or antigen binding
fragment thereof comprises a heavy chain variable domain comprising
the amino acid sequence of SEQ ID NO: 3 and a light chain variable
domain comprising the amino acid sequence of SEQ ID NO: 5.
100. The method of claim 99, wherein the antibody comprises (i) a
heavy chain comprising the same amino acid sequence as the amino
acid sequence of the heavy chain encoded by the plasmid deposited
with the American Type Culture Collection (ATCC) as PTA-10772 and
(ii) a light chain comprising the same amino acid sequence as the
amino acid sequence of the light chain encoded by the plasmid
deposited with the ATCC as PTA-10774.
101. The method of claim 97, wherein a FOLR1 staining intensity
score of 2 or greater has been detected in greater than 75% of the
cells in a tumor sample from the human subject using the IHC
detection method.
102. The method of claim 101, wherein: (A) comprises an antibody or
antigen binding fragment thereof comprising: a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO: 3 and a
light chain variable domain comprising the amino acid sequence of
SEQ ID NO: 5, (L) comprises the linker N-succinimidyl
4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), and (C)
comprises the cytotoxic agent
N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine
(DM4), and wherein the linker (L) links (A) to (C).
103. The method of claim 102, wherein the antibody comprises (i) a
heavy chain comprising the same amino acid sequence as the amino
acid sequence of the heavy chain encoded by the plasmid deposited
with the American Type Culture Collection (ATCC) as PTA-10772 and
(ii) a light chain comprising the same amino acid sequence as the
amino acid sequence of the light chain encoded by the plasmid
deposited with the ATCC as PTA-10774.
104. The method of claim 97, further comprising detecting FOLR1
expression in the tumor sample from the human subject using the IHC
detection method prior to administering the therapeutically
effective dose of the anti-FOLR1 immunoconjugate to the human
subject.
105. The method of claim 97, wherein the tumor sample is a formalin
fixed paraffin embedded sample.
106. The method of claim 97, wherein the IHC detection method
comprises detecting FOLR1 expression with a detection antibody or
antigen binding fragment thereof that specifically binds FOLR1,
wherein the detection antibody or antigen binding fragment thereof
comprises a detection reagent selected from the group consisting
of: an enzyme, a fluorophore, a radioactive label, and a
luminophore.
107. The method of claim 106, wherein the detection reagent is
selected from the group consisting of: biotin, digoxigenin,
fluorescein, tritium, and rhodamine.
108. The method of claim 97, wherein a staining intensity score of
3 or greater for FOLR1 expression has been detected in greater than
75% of cells of the tumor sample.
109. A method for treating ovarian cancer comprising: (a)
contacting a tumor tissue sample from a human subject having
ovarian cancer with a detection antibody or antigen binding
fragment thereof that specifically binds FOLR1, wherein the sample
is formalin-fixed paraffin embedded; (b) measuring the binding of
the detection antibody or antigen binding fragment thereof to FOLR1
in the tumor tissue sample in step (a) using an IHC detection
method that can distinguish between staining intensity and staining
uniformity in the FOLR1 expressing tumor tissue sample as compared
to staining intensity or staining uniformity in one or more
reference samples; (c) assigning a FOLR1 expression score to the
tumor tissue sample after comparing the level of FOLR1 staining
intensity and staining uniformity in the tumor tissue sample to one
or more reference samples; and (d) administering an anti-FOLR1
immunoconjugate to the human subject whose tumor tissue sample has
greater than 25% of cells having a FOLR1 staining intensity score
of 2 or greater.
110. The method of claim 109, wherein the tumor tissue sample has
greater than 75% of cells having a FOLR1 staining intensity score
of 2 or greater.
111. The method of claim 109, wherein the anti-FOLR1
immunoconjugate has the formula (A)-(L)-(C), wherein: (A) comprises
an antibody or antigen binding fragment thereof comprising: (a) a
heavy chain CDR1 comprising the amino acid sequence GYFMN (SEQ ID
NO:6); a heavy chain CDR2 comprising the amino acid sequence
RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising
the amino acid sequence YDGSRAMDY (SEQ ID NO:8); and (b) a light
chain CDR1 comprising the amino acid sequence KASQSVSFAGTSLMH (SEQ
ID NO:9); a light chain CDR2 comprising the amino acid sequence
RASNLEA (SEQ ID NO:10); and a light chain CDR3 comprising the amino
acid sequence QQSREYPYT (SEQ ID NO:11), (L) comprises the linker
N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB),
and (C) comprises the cytotoxic agent
N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine
(DM4), and wherein the linker (L) links (A) to (C).
112. The method of claim 111, wherein the antibody or antigen
binding fragment thereof of the immunoconjugate comprises a heavy
chain variable domain comprising the amino acid sequence of SEQ ID
NO: 3 and a light chain variable domain comprising the amino acid
sequence of SEQ ID NO: 5.
113. The method of claim 111, wherein the antibody of the
immunoconjugate comprises (i) a heavy chain comprising the same
amino acid sequence as the amino acid sequence of the heavy chain
encoded by the plasmid deposited with the ATCC as PTA-10772 and
(ii) a light chain comprising the same amino acid sequence as the
amino acid sequence of the light chain encoded by the plasmid
deposited with the ATCC as PTA-10774.
114. The method of claim 109, wherein the tumor tissue sample is a
formalin fixed paraffin embedded sample.
115. The method of claim 109, wherein the tumor tissue sample has
greater than 75% of cells having a staining intensity score of 3 or
greater.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/807,831, filed on Nov. 9, 2017 (now U.S. Pat. No.
11,135,305), which is a continuation of U.S. application Ser. No.
14/245,797, filed on Apr. 4, 2014 (abandoned), which is a
continuation of U.S. application Ser. No. 13/435,857, filed on Mar.
30, 2012 (now U.S. Pat. No. 8,709,432), which claims the benefit of
U.S. Provisional Appl. No. 61/471,007, filed Apr. 1, 2011, each of
which is herein incorporated by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence listing
in ASCII text file (Name: 2921_0150004_Seqlisting_ST25.txt; Size:
8,302 bytes; and Date of Creation: Aug. 31, 2021), filed with the
application, is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] 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
specifically, the invention concerns more effective treatment of
patients susceptible to or diagnosed with cancer, in which the
tumor cells overexpress FOLR1 as determined by a gene expression
assay, with a FOLR1 antagonist, e.g., a FOLR1 immunoconjugate.
Background Art
[0004] 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 lifetime. 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).
[0005] Folate Receptor 1 (FOLR1), also known as Folate
Receptor-alpha, or Folate Binding Protein, is an N-glycosylated
protein expressed on plasma membrane of cells. FOLR1 has a high
affinity for folic acid and for several reduced folic acid
derivatives. FOLR1 mediates delivery of the physiological folate,
5-methyltetrahydrofolate, to the interior of cells.
[0006] FOLR1 is overexpressed in vast majority of ovarian cancers,
as well as in many uterine, 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 (Weitman S D, et al., Cancer
Res 52: 3396-3401 (1992); Antony A C, Annu Rev Nutr 16: 501-521
(1996); Kalli K R, et al. Gynecol Oncol 108: 619-626 (2008)). This
expression pattern of FOLR1 makes it a desirable target for
FOLR1-directed cancer therapy.
[0007] 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 available procedures,
typically chemotherapeutic drugs after surgical 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 V N et al., Obstet
Gynecol Surv 64: 548-560 (2009)). Thus, there is a clear unmet
medical need for more effective therapeutics for ovarian
cancers.
SUMMARY OF THE INVENTION
[0008] The present invention is based on the discovery of a dynamic
range of expression of FOLR1 in tumor tissue and the discovery that
tumors with increased levels of FOLR1 expression are more
responsive to treatment with anti-FOLR1 antibodies or anti-FOLR1
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-FOLR1
antibodies or anti-FOLR1 immunoconjugates, to patients who are
found to have an increased expression level of FOLR1.
[0009] The present invention provides a method for identifying a
subject pre-disposed to respond favorably to a Folate Receptor 1
(FOLR1)-targeting anti-cancer therapeutic, the method comprising
detecting FOLR1 expression in a tissue sample from the subject.
[0010] 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
FOLR1-targeting anti-cancer therapeutic to a subject, wherein FOLR1
expression in a tissue sample from the subject has been found to be
increased.
[0011] The present invention also provides a method for predicting
effectiveness of a low-dose cancer treatment, the method comprising
administering a therapeutically effective dose of a FOLR1-targeting
anti-cancer therapeutic to a subject, wherein said subject has been
found to have increased expression of FOLR1 in a sample.
[0012] In one embodiment, the methods are directed to ovarian
carcinoma, non-small cell lung adenocarcinoma (including
bronchioloalveolar carcinoma), renal carcinomas, and endometrial
carcinomas.
[0013] In one embodiment, the extent and uniformity of FOLR1
expression is detected by immunohistochemistry (IHC), flow
cytometry, or nucleic acid hybridization. In another embodiment,
the level of FOLR1 expression is detected by immunohistochemistry.
Non-limiting examples of IHC include IHC methods that distinguish
between varying levels of FOLR1 and calibrated IHC methods, such as
those described herein. The FOLR1 expression can be scored using an
appropriate scoring system, including but not limited to the
scoring methods described herein. For example, FOLR1 expression can
be scored using a calibrated IHC method that includes a range of 0,
1, 2, 3, and 3+ for staining intensity with 0 being the lowest
level of staining intensity and 3+ being the highest level of
staining intensity. Alternatively or additionally, FOLR1 expression
can be scored using a calibrated 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.
[0014] In a further embodiment, the FOLR1 expression in a sample
(e.g., a tumor tissue sample) is measured and compared to one or
more reference samples and the FOLR1 expression in the tissue
sample from a subject tumor, xenograft tumor, or cell line has a
FOLR1 specific score correlating 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+ FOLR1 staining intensity with a homogeneous staining pattern is
considered to have increased FOLR1 expression; a tissue sample or
cell with a level 3 FOLR1 staining intensity with heterogeneous or
focal staining patterns is considered to have increased FOLR1
expression. In another embodiment the FOLR1 expression in a sample
is measured and compared to one or more reference samples to
identify a comparable level of staining. 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 antigen or
ABC number for the sample tissue can be determined based on the
comparison.
[0015] In one embodiment, the FOLR1 expression in a sample (e.g., a
tumor tissue sample) is measured and compared to one or more
control samples and the FOLR1 expression in the tissue sample from
a subject tumor, xenograft tumor, or cell line has a FOLR1 specific
score correlating to extent and uniformity of expression as
compared to the one or more control samples. In one embodiment, the
FOLR1 expression in the sample is compared to a negative control
sample which demonstrates no or low detectable FOLR1 expression. In
another embodiment, the FOLR1 expression in the sample is compared
to a positive control sample having increased FOLR1 expression
(level 1, 2, 3 or 3+). In some embodiments, the control samples
include, but are not limited to Namalwa, SW2, SW620, T47D, IGROV-1,
300.19 FR1, HeLa, or KB cells. In particular embodiments, the
control samples include cells or cell pellets from cells
transfected with folate receptor (e.g., 300.19 FR1).
[0016] In one embodiment, the FOLR1-targeting anti-cancer
therapeutic is a FOLR1 immunoconjugate. In one embodiment, the
immunoconjugate comprises an anti-FOLR1 antibody, a linker, and a
cytotoxin.
[0017] In a further embodiment, the anti-FOLR1 antibody is huMOV19.
In another embodiment, the linker is selected from the group
consisting of a cleavable linker, a non-cleavable linker, a
hydrophilic linker, and a 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 4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP);
N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) or
N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB);
N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC);
N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate
(sulfoSMCC); N-succinimidyl-4-(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 another embodiment, the cytotoxic agent is selected from the
group consisting of a maytansinoid, maytansinoid analog,
benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocarmycin,
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-oxopropyl)-maytansine or
N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine. In
another embodiment, the cytotoxic agent is
N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine
(DM4). In a further embodiment, the immunoconjugate comprises the
antibody HUMOV19, sulfo-SPDB, and DM4 (IMGN853).
[0018] The invention is also directed to a kit for measuring FOLR1
expression in a subject comprising a FOLR1 detection reagent, and
instructions for use. In one embodiment, the FOLR1 detection
reagent comprises a FOLR1 binding peptide, protein or a molecular
probe (i.e. nucleic acid). In another embodiment, the FOLR1
detection reagent is an anti-FOLR1 antibody. In another embodiment,
the kit further comprises a secondary antibody which binds the
anti-FOLR1 antibody. In one embodiment the antibody is included at
a concentration of 0.5 to 7.5 .mu.g/ml, desirably 0.9 to 3.8+/-0.5
.mu.g/ml. In various embodiments, the antibody is included at a
concentration of 1.0+/-0.5 .mu.g/ml, 1.5+/-0.5 .mu.g/ml, 1.9+/-0.5
.mu.g/ml, 2.5+/-0.5 .mu.g/ml, 3.0+/-0.5 .mu.g/ml, 3.5+/-0.5
.mu.g/ml, 3.8+/-0.5 .mu.g/ml, or up to 4.2 .mu.g/ml. In another
embodiment, the antibody is included in concentrated solution with
instructions for dilutions to achieve a final concentration of 0.9
to 3.8+/-0.5 .mu.g/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, tritium, and rhodamine.
[0019] The kit can also include instructions for detection and
scoring of FOLR1 expression. The kit can also include control or
reference samples. Non-limiting examples of control or reference
samples include tissue samples, cell pellets or cells. The control
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-1, HELA, KB, JEG-3 and cell lines stably or transiently
transfected with an expression vector that expresses FOLR1 (e.g.,
300.19FR1). Exemplary tissues that may be used as normal reference
tissues in the FOLR1 expression detection methods are described
herein and include normal lung, salivary gland, and pancreas.
[0020] The invention is also directed to a method for identifying a
cancer likely to respond to an anti-FOLR1 antibody, or anti-FOLR1
immunoconjugate comprising: (a) contacting a biological sample
comprising cells from said cancer with an agent that binds FOLR1
protein on the cell surface; (b) detecting binding of said agent
that binds FOLR1 protein 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 comparison 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 FOLR1 level that is greater than the score for a normal or
low FOLR1 expressing reference sample or a score for said cancer
FOLR1 level that is equal to or greater than the score for a high
FOLR1 expressing reference sample identifies said cancer as likely
to respond to an anti-FOLR1 antibody or anti-FOLR1 immunoconjugate.
In certain embodiments, the cancer is ovarian or lung cancer.
[0021] The invention is also directed to a method of identifying a
tumor as sensitive to treatment with an anti-FOLR1 antibody, or
anti-FOLR1 immunoconjugate, said method comprising: (a) measuring
the level of FOLR1 expression in a tumor tissue sample obtained
from said tumor, wherein said measuring comprises the use of a
detection method that distinguishes 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; (b) determining a FOLR1 staining intensity score
for said tumor tissue sample; and (c) comparing the FOLR1 staining
intensity score determined in step (b) to a relative value
determined by measuring FOLR1 protein expression in at least one
reference 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-FOLR1 antibody, or anti-FOLR1
immunoconjugate, and wherein a FOLR1 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-FOLR1 antibody, or anti-FOLR1
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 FOLR1
expression.
[0022] The invention is also directed to a method of optimizing a
therapeutic regimen with an anti-FOLR1 antibody or an anti-FOLR1
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 FOLR1; (b)
measuring the binding of said antibody in (a) to said cell surface
FOLR1 in said sample using a detection method that can distinguish
between 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 assigning
a staining score to said sample; and (c) administering a high dose
of an anti-FOLR1 immunoconjugate when the score in step (b) is less
than or equal to the score for a normal or low FOLR1 expressing
reference sample or administering a low dose of an anti-FOLR1
immunoconjugate when the score is greater than the score for a
normal or low FOLR1 expressing reference sample.
[0023] The invention is also directed to a method of detecting the
expression of cell surface FOLR1 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) contacting said sample with an antibody that
specifically 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.
[0024] 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 ovarian or lung cancer
with an agent that binds cell surface FOLR1 protein; (b) detecting
binding of said agent to said biological 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, wherein 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 respond to a low dose
anti-FOLR1 antibody or anti-FOLR1 immunoconjugate. In certain
embodiments, the method further comprises administering a
therapeutically effective amount of a humanized anti-FOLR1 antibody
or an anti-FOLR1 immunoconjugate to said subject.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0025] FIG. 1. Manual Staining Method: Anti-FOLR1 antibodies detect
FOLR1 expression in transfected cells. 300.19 cells were
transfected with a polynucleotide that encodes human FOLR1. FOLR1
protein expression was detected using the murine antibody BN3.2.
Smith A E et al, Hybridoma (Larchmt). 2007 October;
26(5):281-8.
[0026] FIG. 2. Manual Staining Method: Anti-FOLR1 antibodies 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 antibody was approximately 4000
antibodies bound per cell (ABC).
[0027] FIG. 3. Manual Staining Method: Anti-FOLR1 antibodies can
distinguish different levels of FOLR1 expression in tissue samples.
BN3.2 was used to detect FOLR1 expression in both ovarian tumors
(A), as well as non-small cell lung cancer tumors (B).
[0028] FIG. 4. Manual Staining Method: Uniform FOLR1 expression in
ovarian and NSCLC tumors. FOLR1 expression was high in many of the
ovarian carcinomas, as well as lung adenocarcinomas and
bronchioloalveolar carcinomas tested. The majority of ovarian
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.
[0029] FIG. 5. Manual Staining Method: FOLR1 expression is
generally confined to the membrane of NSCLC cells. High resolution
microscopy revealed that the majority of FOLR1 staining was
restricted to the membrane in NSCLC tumors.
[0030] FIG. 6. Manual Staining Method: FOLR1 expression is
generally confined to the membrane of ovarian cancer cells. High
resolution microscopy revealed that the majority of FOLR1 staining
was restricted to the membrane in ovarian tumors.
[0031] FIG. 7. In vivo efficacy of huMov19-targeted conjugates in a
KB xenograft model. FOLR1-targeting cleavable conjugate
huMov19-SPDB-DM4 (B) in comparison with non-FOLR1-targeting
huC242-SPDB-DM4 (D), and non-cleavable conjugate
huMov19-PEG4-Mal-DM4 (C) in comparison with non-targeting
huC242-PEG4Mal-DM4 (E) were tested using an established xenograft
model of KB cells implanted subcutaneous into SCID mice. Targeting
of FOLR1 by huMov19 resulted in significant reduction in mean tumor
volume.
[0032] FIG. 8. Dose-response anti-tumor activity of IMGN853
treatment in OVCAR-3 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.
[0033] FIG. 9. Dose-response anti-tumor activity of IMGN853
treatment in IGROV-1 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.
[0034] FIG. 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.
[0035] FIG. 11. Dose-response anti-tumor activity of IMGN853
treatment in SKOV-3 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.
[0036] FIG. 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 animals received a single
intravenous injection of PBS.
[0037] FIG. 13. Automated Staining Methods: Representative
Photographs and Histograms depicting FOLR1 Expression in Cell Lines
by IHC and Flow Cytometry. SW620, T47D, Igrov-1, 300.19/FR1, HeLa,
and KB cells were all scored for FOLR1 staining intensity and
uniformity. SW630 and IGROV-1 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.
[0038] FIG. 14. Automated Staining Methods: Representative FOLR1
Staining in Serous Ovarian Cancer. Staining patterns demonstrating
3 homo, 2-3 homo, 2 homo, and 2 hetero staining are shown for
tissue sections from serous ovarian cancer by IHC.
[0039] FIG. 15. Automated Staining Methods: Representative FOLR1
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.
[0040] FIG. 16. Automated Staining Methods: Representative FOLR1
Staining in NSCLC of the Adenocarcinoma Subtype (excluding
bronchioloalveolar 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.
[0041] FIG. 17. Automated Staining Methods: Representative FOLR1
Staining in Endometrial Adenocarcinoma. Staining patterns
demonstrating 3 hetero, 2 hetero, and 1 hetero staining are shown
for tissue sections from endometrial adenocarcinoma by IHC.
[0042] FIG. 18. Automated Staining Methods: Representative FOLR1
Staining in Renal Clear Cell Carcinoma. Staining patterns
demonstrating 2 homo, 2 hetero, and 1 heteto staining are shown for
tissue sections from renal cell cancer by IHC.
[0043] FIG. 19. The cytotoxic activity of IMGN853 in vitro. Five
FOLR1-positive cell lines (KB, IGROV-1, JEG-3, SKOV-3 and OVCAR-3)
and two FOLR1-negative cell lines (Namalwa and SW2) were analyzed
for their sensitivity to the cytotoxic effects of IMGN853. Cells
were exposed to IMGN853 (solid line) or to IMGN853 plus 0.5 .mu.M
unconjugated huMov19 (M9346A) (dashed line) for 5 days, and the
cell survival was determined by WST-8-based assay. Representative
data are shown. The percent of surviving cells was plotted against
base 10 logarithm of the concentration of IMGN853.
[0044] FIG. 20. The sensitivity of the FOLR1-positive cell lines to
IMGN853 versus the level of FOLR1 expression. Potency and
specificity of IMGN853 was analyzed against FOLR1-positive cell
lines with a wide range of FOLR1 expression. Cell lines were
incubated wih IMGN853 and KB, Igrov-1, and Jeg-3 were specifically
sensitive to IMGN853 while unconjugated huMov19 (M9346A) showed
decreased activity of the conjugate. Skov-3 and Ovcar-3 were not
sensitive to IMGN853 and unconjugated huMov19 (M9346A) did not
change the activity of the conjugate.
[0045] FIG. 21. Automated Staining Methods: Ovarian Carcinoma
Xenograft efficacy models stained for FOLR1. Staining patterns
demonstrating 1-3 hetero (Ovcar 3), 1-3 homo (Igrov 1), 1-2 hetero
(Ov 90) and negative (SKOV 3) are shown for tissue sections from
ovarian cancer xenografts by IHC.
[0046] FIG. 22. Automated Staining Methods: Mouse Xenograft Models.
Staining patterns for FOLR1 in xenografts for NSCLC (A),
Endometrium Carcinoma (B) and Cervical Carcinoma (C) Cell Lines are
shown. NSCLC samples demonstrated 2-3 homo or 2 homo staining,
endometrium carcinoma demonstrated 2 hetero/3 focal staining, and
cervical carcinoma demonstrated 3 homo staining.
[0047] FIG. 23. Assay control tissues automated staining guide.
Staining patterns for negative (esophagus 0) and positive control
samples (salivary gland 1-2 hetero, lung 2 homo, pancreas 3 homo)
are shown as determined by automated IHC.
[0048] FIG. 24. Tumor tissues automated staining guide.
Representative staining patterns for level 3, level 2, and level 1
staining are shown on control tissue as determined by automated
IHC.
[0049] FIG. 25. Tumor tissues automated staining guide.
Representative staining patterns for level 3, level 2, and level
1/negative staining are shown on control tissue as determined by
automated IHC.
DETAILED DESCRIPTION OF THE INVENTION
[0050] The present invention provides methods for increasing the
efficacy of or the likelihood of response to the treatment of
cancers characterized by the overexpression of FOLR1. The present
invention is based on the discovery of a dynamic range of
expression of FOLR1 in tumor tissue as compared to normal tissue
and the discovery that tumors with increased levels of FOLR1
expression are more responsive to treatment with anti-FOLR1
antibodies or anti-FOLR1 immunoconjugates. We have also discovered
differences in sensitity and detection of dynamic ranges between
automated and manual methods. Kits comprising one or more reagents
useful for practicing the methods of the invention are further
provided.
I. Definitions
[0051] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below.
[0052] The terms "human folate receptor 1" or "FOLR1", as used
herein, refers to any native human FOLR1, unless otherwise
indicated. The term "FOLR1" encompasses "full-length," unprocessed
FOLR1 as well as any form of FOLR1 that results from processing
within the cell. The term also encompasses naturally occurring
variants of FOLR1, e.g., splice variants, allelic variants and
isoforms. The FOLR1 polypeptides described herein can be isolated
from a variety of sources, such as from human tissue types or from
another source, or prepared by recombinant or synthetic methods.
Examples of FOLR1 sequences include, but are not limited to NCBI
reference numbers P15328, NP 001092242.1, AAX29268.1, AAX37119.1,
NP 057937.1, and NP 057936.1, and those shown in SEQ ID NOs: 1 and
2.
[0053] The term "increased expression" of FOLR1 refers to a sample
which contains elevated levels of FOLR1 expression. In one example,
the FOLR1 expression is measured by IHC and given a staining
intensity score or a staining uniformity score by comparison to
controls (e.g., calibrated 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 control 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 FOLR1. A staining uniformity that is
heterogeneous or homogeneous is also indicative of increased FOLR1
expression. The staining intensity and staining 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 FOLR1
expression 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
FOLR1 values).
[0054] A "reference sample" can be used to correlate and compare
the results obtained in the methods of the invention from a test
sample. Reference samples can be cells (e.g., cell lines, cell
pellets) or tissue. The FOLR1 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
FOLR1. The diagnostic methods of the invention involve a comparison
between expression levels of FOLR1 in a test sample and a
"reference value." In some embodiments, the reference value is the
expression level of the FOLR1 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 reference value can be a single
cut-off value, such as a median or mean or a range of values, such
as a confidence interval. Reference values can be established for
various subgroups of individuals, such as individuals predisposed
to cancer, individuals having early or late stage cancer, male
and/or female individuals, or individuals undergoing cancer
therapy. Examples of normal reference samples or values and
positive reference samples or values are described herein.
[0055] In some embodiments, the reference sample is a sample from a
healthy tissue, in particular a corresponding tissue which is not
affected by cancer. These types of reference samples are referred
to as negative control samples. In other embodiments, the reference
sample is a sample from a tumor tissue that expresses FOLR1. 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 staining intensity, which correlates
with the level of FOLR1 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 FOLR1-expressing reference samples
include human esophagus tissue; low FOLR1 reference includes
salivary gland (particularly the intercalated ducts) and lung
(particularly respiratory epithelium) tissue; and high
FOLR1-expressing tissue includes the pancreas (particularly ductal
cells). For cell lines, low expressors include, but are not limited
to OVCAR3 and T47D, moderate expressers include, but are not
limited to SW620, IGROV-1, JEG3, and high expressers include, but
are not limited to, KB and IGROV1. Particularly desirable positive
high FOLR1 reference is a cell line stably or transiently
transfected with Folate Receptor 1 (e.g., 300.19/FR1). Appropriate
positive and negative reference levels of FOLR1 for a particular
cancer, may be determined by measuring levels of FOLR1 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
FOLR1 levels in samples from subjects of a certain age and
reference levels for a particular disease state, phenotype, or lack
thereof in a certain age group). Such reference levels may also be
tailored to specific techniques that are used to measure levels of
FOLR1 in biological samples (e.g., immunoassays, etc.), where the
levels of FOLR1 may differ based on the specific technique that is
used.
[0056] 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 generally the first antibody used in
an immunohistochemical (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
between the primary antibody and a subsequent reagent, if any. The
secondary antibody is generally the second antibody used in an
immunohistochemical procedure.
[0057] 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, tumors, organs, cell
cultures and/or cell culture constituents, for example. The present
invention is particularly useful for cancer samples which generally
comprise 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 FOLR1 or a
state of a sample, including, but not limited to, comparing
different types of cells or tissues, comparing different
developmental stages, and detecting or determining the presence
and/or type of disease or abnormality.
[0058] 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
selected portion can be as small as one cell or two cells, or could
represent many thousands of cells, for example. In most cases, the
collection of cells is important, and while the invention has been
described for use in the detection of cellular components, the
method may also be used for detecting non-cellular components of an
organism (e.g. soluble components in the blood as a non-limiting
example).
[0059] By "correlate" or "correlating" is meant comparing, in any
way, the performance and/or 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 FOLR1
correlates with increased likelihood of effectiveness of a
FOLR1-targeting anti-cancer therapy.
[0060] The term "antibody" means an immunoglobulin molecule that
recognizes and specifically binds to a target, such as a protein,
polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing through at least one antigen
recognition site within the variable region of the immunoglobulin
molecule. As used herein, the term "antibody" encompasses intact
polyclonal antibodies, intact monoclonal antibodies, 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 antibodies, human
antibodies, fusion proteins comprising 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 desired biological activity. An antibody can
be of any the five major classes of immunoglobulins: IgA, IgD, IgE,
IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2), based on the identity of their
heavy-chain constant domains referred to as alpha, delta, epsilon,
gamma, and mu, respectively. The different classes of
immunoglobulins have different and well known subunit structures
and three-dimensional configurations. Antibodies can be naked or
conjugated to other molecules such as toxins, radioisotopes,
etc.
[0061] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
binds, such as FOLR1. In a certain embodiment blocking antibodies
or antagonist antibodies substantially or completely inhibit the
biological activity of the antigen. Desirably, the biological
activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or
even 100%.
[0062] The term "anti-FOLR1 antibody" or "an antibody that binds to
FOLR1" refers to an antibody that is capable of binding FOLR1 with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting FOLR1. The extent
of binding of an anti-FOLR1 antibody to an unrelated, non-FOLR1
protein is less than about 10% of the binding of the antibody to
FOLR1 as measured, e.g., by a radioimmunoassay (MA). In certain
embodiments, an antibody that binds to FOLR1 has a dissociation
constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, or .ltoreq.0.1 nM. Examples of anti-FOLR1 antibodies
are known in the art and are disclosed in US Appl. Pub. No.
2012/0009181, which is herein incorporated by reference.
[0063] 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', F(ab')2, and Fv
fragments, linear antibodies, single chain antibodies, and
multispecific antibodies formed from antibody fragments.
[0064] A "monoclonal antibody" refers to a homogeneous antibody
population involved in the highly specific recognition and binding
of a single antigenic determinant, or epitope. This is in contrast
to polyclonal antibodies that typically include different
antibodies directed against different antigenic determinants. The
term "monoclonal antibody" encompasses both intact and full-length
monoclonal antibodies as well as antibody fragments (such as Fab,
Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins
comprising an antibody portion, and any other modified
immunoglobulin molecule comprising an antigen 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.
[0065] 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 antigen 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, whereas epitopes formed by tertiary folding are
typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation.
[0066] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic 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 variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative
embodiments are described in the following.
[0067] "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 affinity for an antigen of "0.6 nM or
better", the antibody'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.
[0068] 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 between the two values to be of
little or no biological and/or statistical 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.
[0069] A polypeptide, antibody, polynucleotide, vector, cell, or
composition which is "isolated" is a polypeptide, antibody,
polynucleotide, vector, cell, or composition which is in a form not
found in nature. 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,
polynucleotide, vector, cell, or composition which is isolated is
substantially pure.
[0070] 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.
[0071] The term "immunoconjugate" or "conjugate" as used herein
refers to a compound or a derivative thereof that is linked to a
cell binding agent (i.e., an anti-FOLR1 antibody or fragment
thereof) and is defined by a generic formula: C-L-A, wherein
C=cytotoxin, L=linker, and A=cell binding agent or anti-FOLR1
antibody or antibody fragment. Immunoconjugates can also be defined
by the generic formula in reverse order: A-L-C.
[0072] 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 FOLR1 antibody or a fragment
thereof in a stable, covalent manner. Linkers can be susceptible to
or be substantially resistant to acid-induced 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.
[0073] 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
include, but are not limited to, carcinoma, 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 cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney cancer, liver
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma and various types of head and neck cancers.
[0074] "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.
[0075] The terms "cancer cell," "tumor cell," and grammatical
equivalents refer to the total population of cells derived from a
tumor or a pre-cancerous lesion, including both non-tumorigenic
cells, which comprise the bulk of the tumor cell population, and
tumorigenic stem cells (cancer stem cells). As used herein, the
term "tumor cell" will be modified by the term "non-tumorigenic"
when referring solely to those tumor cells lacking the capacity to
renew and differentiate to distinguish those tumor cells from
cancer stem cells.
[0076] 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 "subject" and "patient" are used
interchangeably herein in reference to a human subject.
[0077] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0078] 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
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. Such
formulation can be sterile.
[0079] An "effective amount" of an antibody as disclosed herein is
an amount sufficient to carry out a specifically stated purpose. An
"effective amount" can be determined empirically and in a routine
manner, in relation to the stated purpose.
[0080] The term "therapeutically effective amount" refers to an
amount of an antibody or other drug effective to "treat" a disease
or disorder in a subject 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 "treating". To the extent the drug can prevent growth and/or
kill existing cancer cells, it can be cytostatic and/or cytotoxic.
In certain embodiments, identification of increased FOLR1 levels
allows for administration of decreased amounts of the
FOLR1-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 periods of time
necessary, to achieve the desired prophylactic result. Typically
but not necessarily, 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.
[0081] 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. 50:1S-10S (2009)). For
example, tumor growth inhibition, molecular marker expression,
serum marker expression, and molecular imaging techniques can all
be used to assess therapeutic efficacy of an anti-cancer
therapeutic. With respect to tumor growth inhibition, according to
NCI standards, a T/C.ltoreq.42% is the 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 treated/Median tumor
volume of the control.times.100.
[0082] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
can be detectable by itself (e.g. radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, can
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0083] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer, regardless of mechanism of action. Classes
of chemotherapeutic agents include, but are not limited to:
alkyating agents, antimetabolites, spindle poison plant alkaloids,
cytoxic/antitumor antibiotics, topoisomerase inhibitors,
antibodies, photosensitizers, and kinase inhibitors.
Chemotherapeutic agents include compounds used in "targeted
therapy" and conventional chemotherapy.
[0084] Terms such as "treating" 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)
prophylactic or preventative measures that prevent and/or slow the
development of a targeted pathologic condition or disorder. Thus,
those in need of treatment include 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 subject is successfully
"treated" for cancer according to the methods of the present
invention if the patient shows one or more of the following:
reduction in 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 recurrence, tumor response, complete response,
partial response, stable disease, progressive disease, progression
free survival (PFS), overall survival (OS), each as measured by
standards set by the National Cancer Institute and the U.S. Food
and Drug Administration for the approval of new drugs. See Johnson
et al, (2003) J. Clin. Oncol. 21(7):1404-1411.
[0085] "Progression free survival" (PFS), also referred to as or
"Time to Tumor Progression" (YIP) indicates the length of time
during and after treatment that the cancer does not grow.
Progression-free survival includes the amount of time patients have
experienced a complete response or a partial response, as well as
the amount of time patients have experienced stable disease.
[0086] "Disease free survival" (DFS) refers to the length of time
during and after treatment that the patient remains free of
disease.
[0087] "Overall Survival" (OS) refers to a prolongation in life
expectancy as compared to naive or untreated individuals or
patients.
[0088] As used in the present disclosure and claims, the singular
forms "a," "an," and "the" include plural forms unless the context
clearly dictates otherwise.
[0089] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0090] The term "and/or" as used in a phrase such as "A and/or B"
herein is intended to include 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 following
embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and
C; A and B; B and C; A (alone); B (alone); and C (alone).
II. Biological Samples
[0091] 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. Cytochem. (1986) 34:1095) are also suitable. The samples
used may also be embedded in paraffin. In one embodiment, the
tissue samples are both formalin-fixed and paraffin-embedded
(FFPE). In another embodiment, the FFPE block is hematoxylin and
eosin stained prior to selecting one or more portions for analysis
in order to select specific area(s) for the FFPE core sample.
Methods of preparing tissue blocks from these particulate specimens
have been used in previous IHC studies of various prognostic
factors, and/or is well known to those of skill in the art (see,
for example, Abbondanzo et al., Am J Clin Pathol. 1990 May;
93(5):698-702; Allred et al., Arch Surg. 1990 January;
125(1):107-13).
[0092] Briefly, any intact organ or tissue may be cut into fairly
small pieces and incubated in various fixatives (e.g. formalin,
alcohol, etc.) for varying periods of time until the tissue is
"fixed". The samples may be virtually any intact tissue surgically
removed from the body. The samples 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
[0093] The present invention further provides antibodies against
FOLR1, 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 covalently bind or complex at least one
desired molecule or moiety. Such a molecule or moiety may be, but
is not limited to, at least one reporter molecule. A reporter
molecule is defined as any moiety that may be detected using an
assay. Non-limiting examples of reporter molecules that have been
conjugated to antibodies include enzymes, radiolabel s, haptens,
fluorescent labels, phosphorescent molecules, chemiluminescent
molecules, chromophores, luminescent molecules, photoaffinity
molecules, colored particles and/or ligands, such as biotin.
[0094] 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 FOLR1 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 variable domain is involved in antibody self-binding
(Kang et al., 1988) and contains epitopes (idiotopes) recognized by
anti-antibodies (Kohler et al., 1989).
[0095] Certain examples of protein binding (e.g., antibody)
conjugates are those conjugates in which the protein binding agent
(e.g., antibody) is linked to a detectable label. "Detectable
labels" are compounds and/or elements that can be detected due to
their specific functional properties, and/or chemical
characteristics, the use of which allows the antibody to which they
are attached to be detected, and/or further quantified if
desired.
[0096] Many appropriate imaging agents are known in the art, as are
methods for their attachment to antibodies (see, for e.g., U.S.
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; radioactive isotopes; fluorochromes; NMR-detectable
substances; and/or X-ray imaging, for example.
[0097] Exemplary fluorescent labels contemplated for use as protein
binding (e.g., antibody) conjugates include Alexa 350, Alexa 430,
Alexa 488, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,
BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,
Dylight 488, Fluorescein Isothiocyanate, 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.
[0098] Protein binding (e.g., antibody) detection conjugates
contemplated in the present invention include 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. Examples of
suitable enzymes include urease, alkaline phosphatase,
(horseradish) hydrogen peroxidase and/or glucose oxidase. Preferred
secondary binding ligands are biotin and/or avidin and streptavidin
compounds. The use of such labels is well known to those of skill
in the art and are described, for example, in U.S. 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.
[0099] 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 particular, 2- and 8-azido analogues of purine
nucleotides have been used as site-directed photoprobes to identify
nucleotide binding proteins in crude cell extracts (Owens &
Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides
have also been used to map nucleotide binding domains of purified
proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et
al., 1989) and may be used as antibody binding agents.
[0100] 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 complex employing, for
example, an organic chelating agent such a
diethylenetriaminepentaacetic acid anhydride (DTPA);
ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;
and/or tetrachloro-3.alpha.-6.alpha.-diphenylglycouril-3 attached
to the antibody (U.S. 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
U.S. Pat. No. 4,938,948, imaging of breast tumors, for example, is
achieved using monoclonal antibodies, and the detectable imaging
moieties are bound to the antibody using linkers such as
methyl-p-hydroxybenzimidate or
N-succinimidyl-3-(4-hydroxyphenyl)-propionate.
[0101] In other embodiments, derivatization of immunoglobulins by
selectively introducing sulfhydryl groups in the Fc region of an
immunoglobulin using reaction conditions that do not alter the
antibody combining site are contemplated. Antibody conjugates
produced according to this methodology are disclosed to exhibit
improved longevity, specificity and sensitivity (U.S. 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 (O'Shannessy
et al., 1987).
[0102] 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)
[0103] The use of substrates and indicators is contemplated for
detection of FOLR1, such as the exemplary embodiments provided
below, for example.
[0104] Horseradish peroxidase (HRP) is an enzyme that first forms a
complex with hydrogen peroxide and then causes it to decompose,
resulting in water and atomic oxygen. Like many other enzymes, HRP
and some HRP-like activities can be inhibited by excess substrate.
The complex formed between HRP and excess hydrogen peroxide is
catalytically inactive and in the absence of an electron donor
(e.g. chromogenic substance) is reversibly inhibited. It is the
excess hydrogen peroxide and the absence of an electron donor that
brings about quenching of endogenous HRP activities.
[0105] 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 admixed with the antibody, resulting in much more efficient
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 biotinyl-N-hydroxysuccinimide
ester or to biotin hydrazide before it can be reacted with the
epsilonamino groups of the HRP enzyme.
[0106] 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 density of polymerized DAB, gold chloride in combination
with silver sulfide appears to be the most successful.
[0107] 3-Amino-9-ethylcarbazole (AEC) is a substrate for enzymes
such as HRP. Upon oxidation, forms a rose-red end product that is
alcohol soluble. Therefore, specimens processed with AEC must not
be immersed in alcohol or alcoholic solutions (e.g., Harris'
hematoxylin). Instead, an aqueous counterstain and mounting medium
should be used. AEC is unfortunately susceptible to further
oxidation and, when exposed to excessive light, will fade in
intensity. Storage in the dark is therefore recommended.
[0108] 4-Chloro-1-naphthol (CN) is a substrate for enzymes such as
HRP and precipitates as a blue end product. Because CN is soluble
in alcohol and other organic solvents, the specimen must not be
dehydrated, exposed to alcoholic counterstains, or coverslipped
with mounting media containing organic solvents. Unlike DAB, CN
tends to diffuse from the site of precipitation.
[0109] p-Phenylenediamine dihydrochloride/pyrocatechol
(Hanker-Yates reagent) is a an electron donor substrate for enzymes
such as HRP and gives a blue-black reaction product that is
insoluble in alcohol and other organic solvents. Like polymerized
DAB, this reaction product can be osmicated. Varying results have
been achieved with Hanker-Yates reagent in immunoperoxidase
techniques.
[0110] 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-0 bond; an
intermediate enzyme-substrate bond is briefly formed. The chief
metal activators for AP are Mg++, Mn++ and Ca++.
[0111] 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 procedure 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 phosphatase activity from bone, kidney, liver and some
white cells can be inhibited by the addition of 1 mM levamisole to
the substrate solution, although 5 mM has been found to be more
effective. Intestinal alkaline phosphatases are not adequately
inhibited by levamisole.
[0112] In the immunoalkaline phosphatase staining method, the
enzyme hydrolyzes naphthol phosphate esters (substrate) to phenolic
compounds and phosphates. The phenols couple to colorless diazonium
salts (chromogen) to produce insoluble, colored azo dyes. Several
different combinations of substrates and chromogens have been used
successfully.
[0113] 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
produce 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 preferred when staining
cell smears.
[0114] Additional exemplary substrates include naphthol AS-BI
phosphate, naphthol AS-TR phosphate and 5-bromo-4-chloro-3-indoxyl
phosphate (BCIP). Other possible chromogens include Fast Red LB,
Fast Garnet GBC, Nitro Blue Tetrazolium (NBT) iodonitrotetrazolium
Violet (INT), and derivatives of the structures, for example.
V. Immunodetection Methods
[0115] In still further embodiments, the present invention concerns
immunodetection methods for binding, purifying, removing,
quantifying and/or otherwise generally detecting biological
components such as a ligand as contemplated by the present
invention. The antibodies prepared in accordance with the present
invention may be employed 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
specific antibodies is contemplated. Some immunodetection methods
include flow cytometry, enzyme linked immunosorbent assay (ELISA),
radioimmunoassay (MA), immunoradiometric assay, fluoroimmunoassay,
chemiluminescent assay, bioluminescent assay, and Western blot to
mention a few. The steps of various useful immunodetection methods
have been described in the scientific literature, such as, e.g.,
Doolittle M H and Ben-Zeev O, Methods Mol Biol. 1999; 109:215-37;
Gulbis B and Galand P, Hum Pathol. 1993 December; 24(12):1271-85;
and De Jager R et al., Semin Nucl Med. 1993 April; 23(2):165-79,
each incorporated herein by reference.
[0116] In general, the immunobinding methods include obtaining a
sample suspected of comprising ligand protein, 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.
[0117] In terms of antigen detection, the biological sample
analyzed 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 FOLR1-containing compositions, or even
any biological fluid that comes into contact with the tissue,
including blood and/or serum, although tissue samples or extracts
are preferred.
[0118] Contacting the chosen biological sample with the antibody
under effective conditions and for a period of time sufficient to
allow the formation of immune complexes (primary immune complexes)
is generally a matter of simply adding the antibody composition to
the sample and incubating the 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 primary immune complexes
to be detected.
[0119] In general, the detection of immunocomplex formation is well
known in the art and may be achieved through the application of
numerous approaches. These methods are generally based upon the
detection of a label or marker, such as any of those radioactive,
fluorescent, biological and enzymatic tags. U.S. Patents concerning
the use of such labels include U.S. 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. Of course, one may find
additional advantages through the use of a secondary binding ligand
such as a second antibody and/or a biotin/avidin ligand binding
arrangement, as is known in the art.
[0120] 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 antibody. In these
cases, the second binding agent 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 detection
system. The primary immune complexes are contacted with the
labeled, secondary binding agent, or antibody/polymer detection
system, under effective conditions and for a period of time
sufficient to allow the formation of secondary immune complexes.
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.
[0121] Further methods include the detection of primary immune
complexes by a two-step approach. A second binding agent, such as
an antibody, that has binding affinity for the antibody is used to
form secondary immune complexes, as described above. After washing,
the secondary immune complexes are contacted with a third binding
agent or antibody that has binding affinity for the second
antibody, again under effective conditions and for a period of time
sufficient to allow the formation of immune complexes (tertiary
immune complexes). The third ligand or antibody is linked to a
detectable label, allowing detection of the tertiary immune
complexes thus formed. This system may provide for signal
amplification if this is desired.
[0122] 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
antigen to form a biotinylated antibody/antigen complex. The
antibody/antigen complex is then amplified by incubation in
successive solutions of streptavidin (or avidin), biotinylated DNA,
and/or complementary 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 with 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.
[0123] Another known method of immunodetection takes advantage of
the immuno-PCR (Polymerase Chain Reaction) 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 suitable primers with appropriate controls. In
specific embodiments, the enormous amplification capability and
specificity of PCR can be utilized to detect a single antigen
molecule. Such detection may take place in real-time. For example,
the use of quantitative real-time PCR is contemplated.
[0124] In the clinical diagnosis and/or monitoring of patients with
various forms of disease, the detection of a FOLR1 mutant, and/or
an alteration in the levels of FOLR1, in comparison to the levels
in a corresponding biological sample from a normal subject is
indicative of a patient 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
background 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.
[0125] In one embodiment, immunological detection (by
immunohistochemistry) of FOLR1 is scored for both intensity and
uniformity (percent of stained cells--membrane only). Comparative
scales for FOLR1 expression for intensity correlate as 0--Negative,
0-1--Very Weak, 1--Weak, 1-2--Weak to Moderate, 2--Moderate,
2-3--Moderate to Strong, 3--Strong. Quantitatively, 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 FOLR1 expression may be characterized as not
overexpressing FOLR1, whereas those samples with 2 or 3 scores may
be characterized as overexpressing FOLR1. Samples overexpressing
FOLR1 may also be rated by immunohistochemical scores corresponding
to the number of copies of FOLR1 molecules expressed per cell, and
have been determined biochemically: 0=0-10,000 copies/cell, 1=at
least about 200,000 copies/cell, 2=at least about 500,000
copies/cell, and 3=at least about 2,000,000 copies/cell.
Comparative scales for FOLR1 percent cell membrane staining
uniformity correlate as follows: 0--Negative, Focal--<25%,
heterogeneous (hetero)--25-75%, and homogeneous
(homo)-->75%.
VI. Nucleic Acid Hybridization
[0126] In situ hybridization is generally carried out on cells or
tissue sections fixed to slides. In situ hybridization may be
performed by several conventional methodologies (See for e.g.
Leitch et al. In situ Hybridization: a practical guide, Oxford BIOS
Scientific Publishers, Microscopy handbooks v. 27 (1994)). In one
in situ procedure, fluorescent dyes (such as fluorescein
isothiocyanate (FITC) that fluoresces green when excited by an
Argon ion laser) are used to label a nucleic acid sequence 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 light source of a wavelength appropriate for excitation
of the specific fluorochrome used.
[0127] Various degrees of hybridization stringency can be employed.
As the hybridization conditions become more stringent, a greater
degree of complementarity is required between 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 dextran sulfate or raising its
concentration may also increase the effective concentration of
labeled probe to increase the rate of hybridization 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.
[0128] Probes used in nucleic hybridization analysis may be either
RNA or DNA oligonucleotides or polynucleotides and may contain not
only naturally-occurring nucleotides but their analogs, like
digoxygenin 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.
[0129] 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 persons of skill in the art will readily
appreciate 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., Molecular Cloning A Laboratory
Manual, Cold Spring Harbor Press, (1989)).
[0130] Probes may also be generated and chosen by several means
including, but not limited to, mapping by in situ hybridization,
somatic cell hybrid panels, or spot blots of sorted chromosomes;
chromosomal linkage analysis; or cloned and isolated from sorted
chromosome libraries from human cell lines or somatic cell hybrids
with human chromosomes, radiation somatic cell hybrids,
microdissection of a chromosome region, or from yeast artificial
chromosomes (YACs) identified by PCR primers specific 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 plasmid,
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 fragments 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 Laboratory Manual, Cold Spring Harbor Press, (1989)].
[0131] Probes are preferably labeled, such as with a fluorophor,
for example. Examples of fluorophores include, but are not limited
to, rare earth chelates (europium chelates), Texas Red, rhodamine,
fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin,
phycocyanin, or commercially available fluorophors such SPECTRUM
ORANGE.TM. and SPECTRUM GREEN.TM. 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 spatially can be identified by a different color
light or pigment resulting from mixing two other colors (e.g.,
light red+green=yellow) pigment (e.g., blue+yellow=green) or by
using a filter set that passes only one color at a time.
[0132] Probes can be labeled directly or indirectly with the
fluorophor, utilizing conventional methodology known to one with
skill in the art.
VII. Detection Kits and Compositions
[0133] Also provided by the invention are kits for use in the
practice of the present invention as disclosed herein. Such kits
may comprise containers, each with one or more of the various
reagents (typically in concentrated form) utilized 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 degenerate primers for use in
detection of nucleic acid molecules by amplification; and/or
reagents and instrumentation for the isolation (optionally by
microdissection) to support the practice of the invention. 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.
[0134] 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.
[0135] 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 antibody. Detectable
labels that are associated with and/or attached to a secondary
binding ligand are also contemplated. Exemplary secondary ligands
are those secondary antibodies or polymers that have binding
affinity for the first antibody.
[0136] Further suitable immunodetection reagents for use in the
present kits include 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/or all such labels may be suitably
employed in connection with the present invention.
[0137] The kits may further comprise a suitably aliquoted
composition of the wild-type and/or mutant protein, polypeptide
and/or polypeptide, whether labeled and/or unlabeled, as may be
used to prepare a standard curve for a detection assay. The kits
may contain antibody- or polymer-label conjugates 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
lyophilized form.
[0138] 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/or
preferably, suitably 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 include injection and/or
blow-molded plastic containers into which the desired vials are
retained.
[0139] The kits may further comprise one or more therapeutic agents
for the treatment of cancer, such as a FOLR1 immunoconjugate and/or
a chemotherapeutic agent.
[0140] The kit may further comprise an a FOLR1 detection reagent
used to measure FOLR1 expression in a subject comprising a FOLR1
detection reagent, and instructions for use. In one embodiment, the
FOLR1 detection reagent comprises a FOLR1 binding peptide, protein
or a molecular probe (i.e. nucleic acid). In another embodiment,
the FOLR1 detection reagent is an anti-FOLR1 antibody. In another
embodiment, the kit further comprises a secondary antibody which
binds the anti-FOLR1 antibody. In one embodiment the FOLR1-specific
antibody is included at a concentration of 0.5 to 7.5 .mu.g/ml,
preferably 0.9 to 3.8+/-0.5 .mu.g/ml. In another embodiment, the
antibody is included at a concentration of 1.0+/-0.5 .mu.g/ml,
1.5+/-0.5 .mu.g/ml, 1.9+/-0.5 .mu.g/ml, 2.5+/-0.5 .mu.g/ml,
3.0+/-0.5 .mu.g/ml, 3.5+/-0.5 .mu.g/ml, 3.8+/-0.5 .mu.g/ml, or up
to 4.2 .mu.g/ml. In another embodiment, the antibody is included in
concentrated solution with instructions for dilutions to achieve a
final concentration of 0.9 to 3.8+/-0.5 .mu.g/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, tritium, and rhodamine.
[0141] The kit can also include instructions for detection and
scoring of FOLR1 expression. The kit can also include control or
reference samples. Non-limiting examples of control or reference
samples include cell pellets or tissue culture cell lines derived
from normal (normal control) or tumor (positive control) samples.
Exemplary cell lines include KB, NCI-H2110, Igrov-1, Ishikawa,
Jeg-3, Skov-3, Hela, T47D, Caco2, SW620, OAW28, HCC827, Ovcar-8,
and Ovcar-3, Ov-90, other tumor cell lines known to express FOLR1,
and cell lines stably or transiently transfected with an expression
vector that expresses FOLR1. Additional examples for positive
control tissues can also be found in Examples 9-11. The kit 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
cancers, as well as those described in the Examples and in FIGS.
23-25)
VIII. FOLR1-Binding Agents
[0142] Any antibodies that bind FOLR1 can be used in the detection
methods of the present invention. Examples of therapeutically
effective anti-FOLR1 antibodies can be found in US Appl. Pub. No.
US 2012/0009181 which is herein incorporated by reference. The
full-length amino acid (aa) and nucleotide (nt) sequences for FOLR1
are known in the art and also provided herein as represented by SEQ
ID NOs: 1 and 2, respectively. A specifically useful antibody for
detection of FOLR1 is the mouse monoclonal anti-huFOLR1 clone BN3.2
(Leica #NCL-L-FRalpha). An example of a therapeutically effective
anti-FOLR1 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 huMov19 (M9346A)
antibody is encoded by the plasmids deposited with the American
Type Culture Collection (ATCC), located at 10801 University
Boulevard, Manassas, Va. 20110 on Apr. 7, 2010 under the terms of
the Budapest Treaty and having ATCC deposit nos. PTA-10772 and
PTA-10773 or 10774. Examples of FOLR1 immunoconjugates useful in
the therapeutic methods of the invention are provided below.
IX. FOLR1 Immunoconjugates
[0143] The present invention also includes methods for increasing
the efficacy of conjugates (also referred to herein as
immunoconjugates), comprising the anti-FOLR1 antibodies, antibody
fragments, functional equivalents, improved antibodies and their
aspects as disclosed herein, linked or conjugated to a cytotoxin
(drug) or prodrug. Exemplary FOLR1 immunoconjugates can be found in
US Appl. Pub. No. US 2012/0009181, which is herein incorporated by
reference. A particularly effective therapeutic immunoconjugate of
the invention comprises the huMov19 antibody described above.
[0144] Suitable drugs or prodrugs are known in the art. In certain
embodiments, drugs or prodrugs are cytotoxic agents. The cytotoxic
agent used in the cytotoxic conjugate of the present invention can
be any compound that results in 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,
dolastatin and dolastatin analogs including auristatins, tomaymycin
derivatives, leptomycin derivatives, methotrexate, cisplatin,
carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine,
melphalan, mitomycin C, chlorambucil and morpholino doxorubicin. In
certain embodiments, the cytotoxic agents are maytansinoids and
maytansinoids analogs.
[0145] The drug or prodrug can, for example, be linked to the
anti-FOLR1 antibody, such as huMov19, or fragment thereof through a
disulfide bond. The linker molecule or crosslinking agent comprises
a reactive chemical group that can react with the anti-FOLR1
antibody or fragment thereof. In certain embodiments, reactive
chemical groups for reaction with the cell-binding agent are
N-succinimidyl esters and N-sulfosuccinimidyl esters. Additionally
the linker molecule comprises a reactive chemical group, in certain
embodiments a dithiopyridyl group that can react with the drug to
form a disulfide bond. In certain embodiments, linker molecules
include, for example, N-succinimidyl 3-(2-pyridyldithio) propionate
(SPDP) (see, e.g., Carlsson et al., Biochem. J, 173: 723-737
(1978)), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see,
e.g., U.S. Pat. No. 4,563,304), N-succinimidyl
4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) (see US
Publication No. 20090274713), N-succinimidyl 4-(2-pyridyldithio)
pentanoate (SPP) (see, e.g., CAS Registry number 341498-08-6),
2-iminothiolane, or acetylsuccinic anhydride.
[0146] 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 limited to, N-succinimidyl 4-(maleimidomethyl)
cyclohexanecarboxylate (SMCC),
N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproa-
te), which is a "long chain" analog of SMCC (LC-SMCC),
maleimidoundecanoic acid N-succinimidyl ester (KMUA),
.beta.-maleimidopropanoic acid N-succinimidyl ester (BMPS),
.gamma.-maleimidobutyric acid N-succinimidyl ester (GMBS),
.epsilon.-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
N-(.alpha.-maleimidoacetoxy)-succinimide ester (AMAS),
succinimidyl-6-(.beta.-maleimidopropionamido)hexanoate (SMPH),
N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and
N-(p-maleimidophenyl)isocyanate (PMPI),
N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl
iodoacetate (SIA), 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-1-carboxylate (SMCC), sulfo-SMCC,
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS or
succinimidyl-iodoacetate, as described in the literature, to
introduce 1-10 reactive groups (Yoshitake et al, Eur. J. Biochem.,
101:395-399 (1979); Hashida et al, J. Applied Biochem., 56-63
(1984); and Liu et al, Biochem., 18:690-697 (1979)).
[0147] The present invention includes aspects wherein about 2 to
about 8 drug molecules ("drug load"), for example, maytansinoid,
are linked to an anti-FOLR1 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 molecules (e.g., a maytansinoid) that
can be attached to a cell binding agent (e.g., an anti-FOLR1
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, 5.0, 5.1,
5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, 8.0, 8.1). In certain embodiments, the drug is
N.sup.2'-deacetyl-N.sup.T-(3-mercapto-1-oxopropyl)-maytansine (DM1)
or N.sup.2'-deacetyl-N.sup.2'-(4-mercapto-4-methyl-1-oxopentyl)
maytansine (DM4). Thus, in a certain embodiment, the antibody
huMov19 is conjugated to DM1 or DM4. In another embodiment, the
antibody FR-1-21 is conjugated to DM1 or DM4. In another
embodiment, the antibody FR-1-48 is conjugated to DM1 or DM4. In
another embodiment, the antibody FR-1-49 is conjugated to DM1 or
DM4. In another embodiment, the antibody FR-1-57 is conjugated to
DM1 or DM4. In another embodiment, the antibody FR-1-65 is
conjugated to DM1 or DM4.
X. Correlation of FOLR1 Expression and Therapeutic Efficacy
[0148] In certain embodiments, the invention provides a method for
identifying subjects with an increased likelihood for responding to
FOLR1-targeting anti-cancer therapies. The invention is based, in
part, on the discovery that elevated FOLR1 expression levels
correlates with efficacy of FOLR-1-targeting anti-cancer
therapeutics.
[0149] 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 FOLR1 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 FOLR1 expression, preferably a score of 2, 3, or
3+, have an increased likelihood to respond to FOLR-1-targeted
anti-cancer therapies at clinically-relevant doses of FOLR1
immunoconjugates (e.g., 5 mg/kg xenograft dose of a FOLR1
immunoconjugate can approximate a 185 mg/m.sup.2 in patients).
Thus, identification of individuals having an elevated FOLR1 score
would help identify those individuals who might respond to a
clinically relevant dosage. As described in more detail below,
sensitivity to FOLR1 therapeutics correlated with FOLR1 scoring of
2 or higher, especially with level 3 scoring. Moreover, expression
of more uniform levels of FOLR1 provides better correlation with
therapeutic benefit. Thus, a homogeneous staining uniformity is
preferred but combinations of increased staining intensity with
heterogeneous staining uniformity are also indicative of increased
FOLR1 expression. For example, scores of greater than 2 hetero is a
patient selection criterion for treatment with a FOLR1 therapeutic
agent.
[0150] FOLR1 expression analysis also identifies patients in whom
decreased levels of a FOLR1-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 smallest dosage that achieves the desired therapeutic response.
This is specifically important for therapeutics that cause
clinical, and often undesired, side effects. The ability to
recognize those subjects with elevated FOLR1 expression levels
allows for minimization of the dosage of the FOLR-1-targeting
therapeutic, thus decreasing possible side effects, while
maintaining therapeutic efficacy.
[0151] As shown herein, FOLR1 expression scores of 2 hetero or
greater correlate with increased responsiveness to anti-FOLR1
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 recurrence, tumor response, complete response,
partial response, stable disease, progressive disease, progression
free survival (PFS), or overall survival (OS). In certain
embodiments, FOLR1 expression scores of 2 hetero or greater
correlate with increasing PFS, DFS, or OS.
[0152] Kits for use in the detection methods and correlation to
reference/control samples 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 or tumor tissue. Positive and negative reference
samples can be derived from cell lines including SW620, T47D,
IGROV-1, HeLa, KB, JEG-3, other tumor cell lines, and cell lines
stably or transiently transfected with an expression vector that
encodes FOLR1. Normal or tumor tissue samples and tissue culture
cell lines can also be used as a negative control reference
samples. For additional samples, see Examples 9-11 and FIGS.
23-25.
XI. Pharmaceutical Compositions and Therapeutic Methods
[0153] FOLR1-binding agents (including antibodies,
immunoconjugates, and polypeptides) are useful in a variety of
applications including, but not limited to, therapeutic treatment
methods, such as 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 vitro, ex
vivo, or in vivo methods. In certain embodiments, the FOLR1-binding
agent or antibody or immunoconjugate, or polypeptide is an
antagonist of the human FOLR1 to which it binds.
[0154] In certain embodiments, the disease treated with the
FOLR1-binding agent or antagonist (e.g., a huMov19 antibody or
immunoconjugate) is a cancer. In certain embodiments, the cancer is
characterized by tumors expressing folate receptor 1 to which the
FOLR1-binding agent (e.g., antibody) binds.
[0155] The present invention provides for methods of treating
cancer comprising administering a therapeutically effective amount
of a FOLR1-binding 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 cancer, pancreatic cancer,
lung cancer, ovarian cancer, liver cancer, breast cancer, brain
cancer, kidney cancer, prostate cancer, gastrointestinal cancer,
melanoma, cervical cancer, bladder cancer, glioblastoma, and head
and neck cancer. In certain embodiments, the cancer is ovarian
cancer. In certain embodiments, the cancer is lung cancer. In
certain embodiments, the subject is a human.
[0156] The present invention further provides methods for
inhibiting tumor growth using the antibodies or other agents
described herein. In certain embodiments, the method of inhibiting
the tumor growth comprises contacting the cell with a FOLR1-binding
agent (e.g., antibody) in vitro. For example, an immortalized cell
line or a cancer cell line that expresses FOLR1 is 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 cultured in medium to which is added
an FOLR1-binding agent to inhibit tumor growth.
[0157] In some embodiments, the method of inhibiting tumor growth
comprises contacting the tumor or tumor cells with the
FOLR1-binding agent (e.g., antibody) in vivo. In certain
embodiments, contacting a tumor or tumor cell with a FOLR1-binding
agent is undertaken in an animal model. For example, FOLR1-binding
agents can be administered to xenografts expressing one or more
FOLR1s that have been grown in immunocompromised mice (e.g.
NOD/SCID mice) to inhibit tumor growth. In some embodiments, the
FOLR1-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 FOLR1-binding agent is
administered as a therapeutic after the tumorigenic cells have
grown to a specified size.
[0158] In certain embodiments, the method of inhibiting tumor
growth comprises administering to a subject an therapeutically
effective amount of a FOLR1-binding agent. In certain embodiments,
the subject is a human. In certain embodiments, the subject has a
tumor or has had a tumor removed.
[0159] 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.
[0160] In certain embodiments, the invention provides methods of
inhibiting tumor growth using low doses of a FOLR1-binding agent.
The term "low dose" as used herein refers to the therapeutically
effective dose of a FOLR1-binding agent which is less than the
usual or the conventional dose required to produce the therapeutic
effect.
[0161] Thus, in certain embodiments the inventions provides methods
of treating cancer using huMov19 antibody and immunoconjugates. In
certain embodiments, the huMov19 immunoconjugate is
huMov19-SPDB-DM4; huMov19-sulfo-SPP-DM1; huMov19-SPP-DM1; or
huMov19-PEG4-Mal-DM4. In a certain embodiment, the huMov19
immunoconjugate is huMov19-SPDB-DM4, which is also referred to as
IMGN853.
[0162] In certain embodiments, formulations are prepared for
storage and use by combining a purified antibody or agent of the
present invention with a pharmaceutically acceptable vehicle (e.g.
carrier, excipient) (Remington, The Science and Practice of
Pharmacy 20th Edition Mack Publishing, 2000). Suitable
pharmaceutically acceptable vehicles include, but are not limited
to, nontoxic buffers such as phosphate, citrate, and other organic
acids; salts such as sodium chloride; antioxidants including
ascorbic acid and methionine; preservatives (e.g.
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride; benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens, such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight polypeptides (e.g. less than about 10 amino acid
residues); proteins such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; carbohydrates such as monosaccharides,
disaccharides, glucose, mannose, or dextrins; chelating agents such
as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and non-ionic surfactants such as TWEEN or
polyethylene glycol (PEG).
[0163] The pharmaceutical compositions of the present invention can
be administered in any number of ways for either local or systemic
treatment. Administration can be topical (such as to mucous
membranes including vaginal and rectal delivery) such as
transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders; pulmonary (e.g., by
inhalation or insufflation of powders or aerosols, including by
nebulizer; intratracheal, intranasal, epidermal and transdermal);
oral; or parenteral including intravenous, intraarterial,
subcutaneous, intraperitoneal or intramuscular injection or
infusion; or intracranial (e.g., intrathecal or intraventricular)
administration.
[0164] An antibody or immunoconjugate of the invention can be
combined in a pharmaceutical combination formulation, or dosing
regimen as combination therapy, with a second compound having
anti-cancer properties. The second compound of the pharmaceutical
combination formulation or dosing regimen preferably has
complementary activities to the ADC of the combination such that
they do not adversely affect each other. Pharmaceutical
compositions comprising the FOLR1-binding agent and the second
anti-cancer agent are also provided.
[0165] For the treatment of the disease, the appropriate dosage of
an antibody or agent of the present invention depends on the type
of disease to be treated, the severity and course of the disease,
the responsiveness of the disease, whether the antibody or agent is
administered for therapeutic or preventative purposes, previous
therapy, patient's clinical history, and so on all at the
discretion of the treating physician. The antibody or agent can be
administered one time or over a series of treatments lasting from
several days to several months, or until a cure is effected or a
diminution of the disease state is achieved (e.g. reduction in
tumor size). Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient and
will vary depending on the relative potency of an individual
antibody or agent. The administering physician can easily determine
optimum dosages, dosing methodologies and repetition rates. In
certain embodiments, dosage is from 0.01 .mu.g 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
FOLR1-binding agent is given once every two weeks or once every
three weeks. In certain embodiments, the dosage of the antibody or
other FOLR1-binding agent is from about 0.1 mg to about 20 mg per
kg of body weight. The treating physician can estimate repetition
rates for dosing based on measured residence times and
concentrations of the drug in bodily fluids or tissues.
[0166] The combination therapy can provide "synergy" and prove
"synergistic", i.e. the effect achieved 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: (1) 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 together.
[0167] Embodiments of the present disclosure can be further defined
by reference to the following 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
without departing from the scope of the present disclosure.
EXAMPLES
[0168] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application.
[0169] The Folate Receptor-1 (FOLR1) has been reported to be highly
expressed in ovarian tumors and expressed at high to moderate
levels in brain, breast, bladder, endometrioid, lung, pancreatic,
and renal carcinomas. However, the expression of FOLR1 is limited
in normal tissues and includes kidney, lung, choroid plexus,
pancreas, breast, thyroid, ovary, prostate, and lung.
[0170] Methods have been reported that quantify FOLR1 using fresh
frozen tissue homogenates. Whole tissue homogenates cannot
distinguish cytoplasmic from membrane-associated expression and
freshly frozen samples are not amenable in the clinical setting.
However, formalin fixed paraffin embedded (FFPE) samples can be
archived for patients in the clinic.
Example 1
[0171] Immunohistochemical staining of FOLR1 in cell
samples--manual methods Formalin fixed paraffin embedded cell
pellets and tissues were used as test samples with the following
staining reagents and conditions.
IHC Antibodies FFPE Assay Conditions
TABLE-US-00001 [0172] Test Article mouse monoclonal anti-huFOLR1
clone BN3.2 (Leica # NCL-L- FRalpha) ImmunoGen: Prokaryotic
recombinant protein corresponding to 189 amino acids of the
external domain of the folate receptor alpha molecule Control
Article muIgG1 (Coulter) Clone, Cat# 6602872 Secondary Antibody
Biotinylated horse anti-mouse (Vector, Cat# PK-6100)
TABLE-US-00002 Steps Condition Antigen Retrieval Borg (Biocare) pH
9.0 Blocking Steps Avidin/Biotin; peroxide Test or Control Article
2.0 .mu.g/mL Diluent PBS containing 2% horse serum Secondary
Antibody 10 .mu.g/mL Detection System* Avidin-Biotin-peroxidase-
Complex; ABC (Vector Labs) Chromagen DAB (Dako) DAB development
time .gtoreq.5 min
[0173] Formalin-fixed paraffin-embedded (FFPE) patient ovarian
tumor biopsies and ovarian xenograft tumors were stained with the
murine anti-FOLR1 antibody clone BN3.2, (Leica, Cat #NC1-L-FRalpha)
and a control muIgG1 from Coulter. 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 room temperature for 60
minutes with the anti-FOLR1 or control muIgG1 antibody, followed by
30 minutes with biotinylated anti-mouse IgG and 40 minutes with
avidin-biotin-peroxidase complex to detect bound secondary
antibody. Incubation for 5 minutes with DAB (3,3-diaminobenzidine
tetrahydrochloride) resulted in the color signal. Slides were
counterstained with hematoxylin.
[0174] FOLR1 staining intensity and distribution patterns were
scored relative to control IgG staining (non-specific). Intensity
was scored on a scale of 0 to 3 (0=no staining, 1=weak, 2=moderate
and 3=strong) and distribution was scored as focal (<25% of
cells stained), heterogeneous (25-75% of cells stained) and
homogeneous (>75% of cells stained).
[0175] FFPE samples were derived from tumor micro arrays, as well
as human tissue blocks from seven different tumors, as outlined
below.
FFPE Test Samples
TABLE-US-00003 [0176] Human Tumor Micro Arrays (TMAs) Description
Commercial Source Mixed tumor 96 cores from 33 Biomax Cat# MC961
types of cancer Ovarian tumor 64 cores Biochain Cat# T8235725 NSCLC
80 cores Biomax Cat# LC806 Colorectal carcinoma 85 duplicate cores
Biomax Cat# BC000110 Human Tissue Blocks Number Commercial Source
Breast Tumor 4 CHTN Colorectal Carcinoma 4 Ovarian Tumor 4 NSCLC 8
Pancreatic Tumor 1 Renal Tumor 8 SCCHN 14
[0177] The FOLR1 test article, murine anti-FOLR1 clone BN3.2, was
tested to determine binding specificity to the huFOLR1 antigen.
Using the reported IHC staining methods, FFPE sections of 300-19
and 300-19 transfected with huFOLR1 (300-10/FOLR1) cell pellets
were stained and evaluated for FOLR1. The FOLR1 test article
specifically stained 300-19/FOLR1+ cells and returned no staining
in 300-19 cells (3 homo and negative, respectively). These results
demonstrate that clone BN3.2 specifically targets the huFOLR1
antigen. (FIG. 1).
[0178] The BN3.2 antibody was also used to detect FOLR1 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 description 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 evaluated is the score of the test article minus
the score of the respective control article. The ABC level for each
sample was estimated by comparing the staining score to the
calibrated cell pellet controls.
TABLE-US-00004 Intensity (amount of stain) Uniformity (number of
stained cells) 0 Negative 1 Weak 0 Negative 2 Moderate Focal
<25% 3 Strong Heterogeneous (hetero) 25-75%.sup. 3+ Very Strong
Homogeneous (homo) >75%
[0179] Antibodies bound per cell (ABC) values were determined for
the FOLR1-positive tumor cell lines (KB, IGROV1, JEG3, and OVCAR3)
using anti-FOLR1-Phycoerythyrin, BD Quantibrite Beads, and flow
cytometry and were shown to have different ABC values. (FIG. 2).
Staining conditions were optimized for FOLR1 so that the cell
pellets prepared from the FOLR1-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 IGROV1 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 FOLR1 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-1 cells exhibited the next highest ABC value of
260,000, JEG-3 exhibited a lower ABC value or 41,000 ABC, while
OVCAR3 cells showed the lowest ABC value of 4,000. The staining
results and respective ABC values are listed in the table
below.
ABC values and respective staining results for huFOLR1 on cell
lines and respective cell pellets.
TABLE-US-00005 FOLR1 Cell Line ABC Score KB 1,700,000 3+ homo
IGROV1 260,000 3 homo JEG3 41,000 2-3 hetero OVCAR3 4,000 1-2
hetero Additional cell lines, including Capan-1, Jar, Hec-1-A,
Hec-1-B, Ishikawa, NCI H292, BT474EEI, PA-1, OV-90, CaOv-4, CaOv-3,
A2780, Ovcar-5, Ovcar-4, HCT-15, 786-O, NCI H838, NCI H522, NCI
H2110, NCI H1734, NCI H228, and FU.OV-30 were tested and found to
be FOLR10 positive but FOLR1 expression levels and sensitivity to
the anti-FOLR1 immunoconjugate activity varied. For reference
purposes, cell lines having consistent FOLR1 expression and
sensitivity to anti-FOLR1 immunoconjugates are preferred.
[0180] Particularly important was that the IHC method was able to
reliable detected FOLR1 expression in ovarian carcinomas and
non-small cell lung cancer (NSCLC) tissue samples. As shown in FIG.
3, FOLR1 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 FIG. 4, high staining intensity and uniformity of staining was
also observed in ovarian carcinomas, lung adenocarcinomas, and
bronchioloalveloar carcinomas. Furthermore, expression of FOLR1 in
NSCLC samples (FIG. 5) and in ovarian carcinomas (FIG. 6) were
further found to be predominantly localized 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.
Example 2
In Vivo Efficacy of huMov19-PEG4Mal-DM4 and huMov19-SPDB-DM4
Conjugates in Comparison with Similar Non-Targeting Conjugates in a
KB Xenograft Model
[0181] FOLR1-targeting cleavable conjugate huMov19-SPDB-DM4 in
comparison with non-targeting huC242-SPDB-DM4, and non-cleavable
conjugate huMov19-PEG4-Mal-DM4 in comparison with non-targeting
huC242-PEG4Mal-DM4 were tested using an established xenograft model
of KB cells (very high FOLR1 expression, 3+ homozygous by manual
IHC) implanted subcutaneous into SCID mice. Mice were randomized by
body weight into treatment groups and treated either singly (SPDB
conjugates) 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 FIG. 7. The treatments with either
huMov19-SPDB-DM4, or huMov19-PEG4Mal-DM4 resulted 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 Ovarian Carcinoma Xenografts
[0182] The anti-tumor effect of IMGN853 was evaluated in an
established subcutaneous xenograft model of ovarian carcinoma. SCID
mice were inoculated with OVCAR-3 ovarian carcinoma cells
(1.times.10.sup.7 cells/animal) injected subcutaneously into the
right flank. When the tumors reached about 100 mm3 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.times.width.times.height.times.1/2.
[0183] IMGN853 was highly active against OVCAR-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 (FIG.
8). There were complete tumor regressions (CR) in 6/6 mice treated
with IMGN853 at 5.0 mg/kg. There were partial tumor regressions
(PR) in 6/6 mice and CR in 4/6 mice treated with IMGN853 at the 2.5
mg/kg dose level. IMGN853 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 Activity of IMGN853 Treatment in IGROV-1
Human Ovarian Carcinoma Xenografts
[0184] The anti-tumor effect of IMGN853 was evaluated in an
established subcutaneous xenograft model of ovarian carcinoma. SCID
mice were inoculated with IGROV-1 ovarian carcinoma cells
(1.times.10.sup.7 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 calculated with the formula:
length.times.width.times.height.times.1/2.
[0185] IMGN853 was highly active against IGROV-1 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 (FIG.
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 Carcinoma Xenografts
[0186] The anti-tumor effect of IMGN853 was evaluated in an
established subcutaneous xenograft model of ovarian carcinoma. SCID
mice were inoculated with OV-90 ovarian carcinoma cells
(1.times.107 cells/animal) injected subcutaneously into the right
flank. When the tumors reached about 100 mm3 in size (13 days after
tumor cell inoculation), the mice were randomly divided into four
groups (6 animals per group). 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. A control group of animals received PBS administered
intravenously at the same schedule. Tumor growth was monitored by
measuring tumor size twice per week. Tumor size was calculated with
the formula: length.times.width.times.height.times.1/2.
[0187] IMGN853 was active against OV-90 tumors (IHC score of 3
hetero-homo by manual methods) at the 2.5 and 5.0 mg/kg dose
levels, resulting in T/C values of 36 and 18%, respectively (FIG.
10). Two animals had partial tumor regressions in the 5.0 mg/kg
group; there were no other tumor regressions in any of the
treatment groups. IMGN853 was inactive at the 1.2 mg/kg dose
(T/C=77%).
Example 6
Dose-Response Anti-Tumor Activity of IMGN853 Treatment in SKOV-3
Human Ovarian Carcinoma Xenografts
[0188] 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.times.10.sup.7 cells/animal) injected 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.times.width.times.height.times.1/2.
[0189] IMGN853 was inactive against SKOV-3 tumors (IHC score of 1-3
focal by manual methods) at all doses, with growth of
IMGN853-treated tumors paralleling the PBS control group (FIG. 11).
There was no data analysis performed, and the study was terminated
early based on the inactivity of IMGN853 in this model.
Example 7
Dose-Response Anti-Tumor Activity of IMGN853 Treatment in KB Human
Cervical Adenocarcinoma Xenografts
[0190] The anti-tumor effect of IMGN853 was evaluated in an
established subcutaneous xenograft cervical adenocarcinoma model.
SCID mice were inoculated with KB cervical adenocarcinoma cells
(1.times.10.sup.7 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 1.0, 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.times.width.times.height.times.1/2.
[0191] 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 (FIG. 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 CRs, 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
Immunohistochemical Staining of FOLR1 in Formalin Fixed Paraffin
Embedded (FFPE) Samples-Automated Methods
[0192] The IHC staining assay uses IVD class I reagents including
the Novocastra FOLR1 antibody (Novocastra/Leica Cat #NCI-L-FRalpha,
clone BN3.2) as the test article and the Leica Bond RX automated
stainer. Bound test or control article were detected by incubation
with the Leica Bond Refine detection system which includes a post
primary reagent (rabbit anti-mouse IgG), followed by a polymer
reagent (goat anti-rabbit polymer) and 3,3-Diaminobenzidine
tetrahydrochloride (DAB) chromogen. FFPE samples were stained with
the specified concentration(s) of primary antibody (prepared by
diluting in Leica diluent FOLR1) as outlined below.
[0193] IHC Antibodies
TABLE-US-00006 Test article.sup.i) Folate Receptor Alpha
(Novocastra/Leica Cat # NCI-L-FRalpha, Lot 159506), murine, clone
BN3.2, liquid concentrate: 75 .mu.g/mL Control article.sup.i) IgG1
(Beckman Coulter Cat. # 6602872, Lots2S7SPS04-23 and 2S7SPS04-26)
stock concentration: 1 mg/mL, murine, clone 2T8-2F5
FFPE Assay Method using Leica Bond RX
TABLE-US-00007 Step.sup.i) Action Time Bake Temperature: 60.degree.
C. 30 minutes Dewax Bond Dewax Solution Fixed 100% Ethanol Fixed
Antigen Retrieval Bond ER2 20 minutes Endogenous Peroxide (Refine
kit component) 5 minutes Peroxidase Block Test Article FOLR1 at 1.9
.mu.g/mL in Leica 15 minutes diluent Detection Post Primary Reagent
(Refine 8 minutes kit) Polymer (Refine kit) 8 minutes Mixed DAB
(Refine kit) 10 minutes Counterstain Hematoxylin (Refine kit) 5
minutes
[0194] 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 intensity and distribution
patterns were scored relative to control IgG staining
(non-specific). Intensity was scored on a scale of 0 to 3 (0=no
staining, 1=weak, 2=moderate and 3=strong) and distribution was
scored as focal (<25% of cells stained), heterogeneous (25-75%
of cells stained) and homogeneous (>75% of cells stained). In
normal tissue, only the defined substructures were evaluated when
calculating intensity and proportion.
IHC Scoring System Consisting of Intensity and Uniformity
Scales
TABLE-US-00008 [0195] Intensity Observed Intensity Category
Intensity Reported Intensity (brightness of stain) 0 Negative 0 0-1
Very Weak 1 1 Weak 1-2 Weak to Moderate 2 2 Moderate 2-3 Moderate
to Strong 3 3 Strong Uniformity (percent of stained cells-membrane
only) 0 Negative Focal <25% Heterogeneous (hetero) 25-75%
Homogeneous (homo) >75%
[0196] FFPE tumor samples were derived from tumor micro arrays, as
well as human tissue blocks from seven different tumors, as
outlined below.
FFPE Test Samples: TMAs
TABLE-US-00009 [0197] Number of Total Anatomic Cores per # of Site
Vendor Catalog # Code Patient Patients Kidney Pantomics KIC1501
P-T-ARR- 2 69 KID- 122711-1 Lung Pantomics LUC1501 P-T-ARR- 2 70
LNG- 122711-1 Lung Tristar 69571059/ P-T-ARR- 1 110 TA1249 OVA-
122711-1 Ovary Biochain T8235725-5 P-T-ARR- 1 62 OVA- 122111-1
Ovary Pantomics OVC1501 P-T-ARR- 2 70 OVA- 122711-1 Ovary Tristar
69571091/ P-T-ARR- 2 96 TA1322 OVA- 010912-1 Uterus Pantomics
EMC1501 P-T-ARR- 2 70 (Endo- EME- metrium) 122711-1 Various
Pantomics MTU481 P-T-ARR- 1 48 122711-1
FFPE Test Samples: Whole Sections
TABLE-US-00010 [0198] Organ Code Source Diagnosis (per Source
Documentation) Ovary 1 Unknown endometroid adenocarcinoma 2
Proteogenex endometroid adenocarcinoma 3 CHTN adenocarcinoma, mixed
with features of endometriod, serous and clear 4 CHTN
adenocarcinoma, high grade w/mixed papillary, serous, endometrioid
and clear cell areas 5 CHTN serous papillary adenocarcinoma 6
Proteogenex serous adenocarcinoma 7 Proteogenex serous papillary
adenocarcinoma 8 Proteogenex serous papillary adenocarcinoma 9 CHTN
serous papillary adenocarcinoma 10 Proteogenex serous papillary
adenocarcinoma Lung 1 CHTN adenocarcinoma, poorly differentiated 2
CHTN adenocarcinoma, acinar, well differentiated with
bronchioloalveolar features 3 CHTN adenocarcinoma, mucinous
features 4 CHTN adenocarcinoma 5 CHTN adenocarcinoma 6 CHTN
adenocarcinoma (bronchioloalveolar) carcinoma 7 CHTN adenocarcinoma
8 CHTN adenocarcinoma, moderately differentiated, with clear cell
features 9 CHTN adenocarcinoma 10 CHTN squamous cell carcinoma
[0199] Cells (tumor cells or transfected cells) were formalin fixed
and paraffin embedded (FFPE). FFPE cell pellet samples shown to
exhibit varying ranges of FOLR1 expression by flow cytometry and
normal human tissues were used in this study to characterize
positive and negative controls and for analysis of specificity. The
cell pellets exhibiting varying levels of FOLR1 and the respective
scores are reported below. There is a poor correlation between
staining scores and the respective FOLR1 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 IGROV-1 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 Cell Pellets at a Test Article Concentration of
1.9 .mu.g/mL
TABLE-US-00011 Cell Line .sup.aABC Value Staining Score SW620
40,098 1-3 hetero T47D 97,576 1-2 hetero IGROV-1 565,481 1-3 hetero
300.19/FR1 830,003 3 homo HELA 1,500,587 2-3 hetero KB 4,000,000 3
homo .sup.aThe reported ABC Value is an average of antibodies bound
per cell in the cell population and was determined as follows: a
concentration of 1.0 .times. 10.sup.-8 M of anti-FOLR1-PE (1:1) was
used to determine ABC values on the respective cell line using flow
cytometry methods and Quantibrite .TM. Beads (BD Biosciences).
[0200] The flow cytometry histograms represent the distribution of
cells versus the number of anti-FOLR1 bound per cell (FOLR1
expression level). Both the histograms and respective IHC staining
results indicate that each of these cells lines contain a
heterogeneous population of cells having a broad range of FOLR1
expression. The exception is the 300.19/FR1 cell line showing both
a uniform flow cytometry histogram and IHC staining score. This
data suggests that cell lines each expressing a more uniform level
of FOLR1 may provide a better correlation between ABC values and
respective staining scores. Although the assay demonstrated
positive staining in all FOLR1-positive cell pellet controls, there
is a poor correlation between staining scores and the respective
FOLR1 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 histograms depicting FOLR1 expression in cell lines by IHC and
flow cytometry are shown in FIG. 13
[0201] 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 performed on
a panel of FFPE samples including FOLR1-positive cell pellets and a
TMA consisting of FOLR1 positive and negative normal tissues
(adrenal (cortex/medulla), breast (ducts and lobules/connective
tissue), fallopian tube (surface epithelium/muscle wall), kidney
(tubules/glomeruli), lung (type I/II pneumocytes/interalveolar
connective tissues), pancreas (ducts/islets of Langerhans),
salivary gland (ducts/stroma), skin (eccrine glands/epidermis),
stomach (surface epithelium/submucosa)), and whole sections of
tumor tissues (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 .mu.g/mL) or
control article concentration of 1.9 .mu.g/mL or 3.8 .mu.g/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
staining in negative tissue controls stained with test article and
3) differentiated between varying levels of membrane-associated
FOLR1 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 concentration of 1.9 .mu.g/mL showed the best
dynamic range in staining results using the Leica Bond RX automated
protocols (Bake and Dewax Protocol, HIER using the ER2 for 20
minutes protocol, and the staining protocol IHC F-With Extra
Rinses).
Example 9
Identification and Characterization of Controls that Characterize
the Dynamic Range of the Assay-Automated Staining Methods
[0202] Quality controls: Human normal salivary gland, lung and
pancreas were identified as positive tissue controls to be employed
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 controls which cover the dynamic range of the assay, a
tissue microarray (TMA) consisting of several FOLR1 positive and
negative normal tissue samples 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 3 homo
apical); intercalated ducts of normal human salivary gland (score
of 1-2 hetero); and normal human esophagus (score of 0). Over a
total of 4 assay runs, the identified suitable assay controls from
this TMA gave identical results. These results indicate that the
selected controls give consistent results and span the dynamic
range of the assay.
Structures in Normal Tissues Identified as Controls that Span the
Dynamic Range of the Assay
TABLE-US-00012 [0203] Normal Staining Score Staining Score Organ
Sub-structure (control article) (test article) Esophagus All
structures 0 (negative) 0 (negative) Salivary Intercalated ducts 0
(negative) 1-2 hetero Gland Lung Respiratory 0 (negative) 2 homo
Epithelium Pancreas Ducts 0 (negative) 3 homo apical
Apical Staining is Defined as Polarized Non-Uniform Membrane
Staining
Example 10
Performance Analysis of the Automated Staining Method
[0204] The intended use of this assay is to specifically detect
FOLR1 reproducibly and with the appropriate sensitivity to
differentiate varying levels and varying uniformity of
membrane-associated FOLR1 expression (optimal dynamic range) in
ovarian, endometrial, NSCLC, and kidney FFPE tumor tissues.
Therefore, specificity, reproducibility, and sensitivity were
considered as performance criteria.
[0205] The specificity and sensitivity of the study assay was
evaluated by comparison of normal tissue staining with the study
assay to previously reported results. Staining results from this
study were compared with corresponding staining results from Scorer
et al 2010 (A Full Immunohistochemical Evaluation of a Novel
Monoclonal Antibody to Folate Receptor--alpha. The Novocastra
Journal of Histopathology, REAGENTS: 2010(3):8-12, describing the
same antibody clone BN3.2) with FFPE normal tissue and from the
Tissue Cross Reactivity (TCR) study using IMGN853 (huMov19 (M9346A)
antibody) on fresh frozen normal tissue (ImmunoGen Report
IMH28-003). 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 sensitive methods (study assay and TCR
assay) only. There were no examples of positive staining in the
least sensitive 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 FOLR1 expression in normal tissues.
[0206] 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 consistently localized to the tumor tissue
with normal adjacent tissue components including stroma, 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
suggesting 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 FOLR1
expression in ovarian, endometrial, NSCLC, and kidney FFPE tumor
tissues. Representative photos of serous ovarian, endometroid
ovarian, NSCLC, endometrial carcinoma, and renal clear cell
carcinoma are provided in FIGS. 14-18. Additional representatitive
photos useful, for example, in a staining guide or diagnostic kit,
are shown in FIGS. 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 Subtypes of Ovarian
Tumors
TABLE-US-00013 [0207] Sample Number (%) Any .gtoreq.3 .gtoreq.2
.gtoreq.1 1-3 Posi- Nega- Subtype Total hetero.sup.a hetero.sup.a
hetero.sup.a focal tivity tive Endo- 35 15 18 20 6 26 9 metrioid
(100) (43) (51) (57) (17) (74) (26) Mucinous 29 2 4 5 0 5 24 (100)
(7) (14) (17) (0) (17) (83) Serous 129 44 92 92 8 100 29 (100) (34)
(71) (71) (6) (78) (22) .sup.i)Focal staining patterns were
excluded
Summary of Staining Scores for NSCLC Tumors
TABLE-US-00014 [0208] Sample Number (%) .gtoreq.3 .gtoreq.2
.gtoreq.1 1-3 Any Type Subtype Total hetero.sup.i) hetero.sup.a
hetero.sup.a focal Positivity Negative Adeno- All.sup.b 67 17 39 42
5 47 20 carcinoma (100) (25) (58) (63) (7) 70 30 Specified 7 2 5 5
0 5 2 Bronchiolo- (100) (29) (71) (71) (0) (71) (29) alveolar
Squamous All 74 1 4 6 6 12 62 cell (100) (1) (5) (8) (8) (16) (84)
carcinoma .sup.i)Focal staining patterns were excluded All
adenocarcinoma samples were included except the specified
bronchioloalveolar carcinoma samples
Summary of Staining Scores for Adenocarcinoma of the Endometrium
and Clear Cell Tumors of the Kidney
TABLE-US-00015 [0209] Sample Number (%) Any Tumor/ .gtoreq.3
.gtoreq.2 .gtoreq.1 1-3 Posi- Nega- Subtype Total hetero.sup.a
hetero.sup.a hetero.sup.a focal tivity tive Endo- 58 5 23 30 10 40
18 metrium/ (100) (9) (40) (52) (17) (69) (31) Adeno- carcinoma
Kidney/ 34 0 9 23 6 29 5 Clear (100) (0) (26) (68) (18) (85) (15)
Cell Focal staining patterns were excluded
[0210] The precision of the study assay was investigated 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 stained 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
reproducibility across tissue types with low, medium and high level
of expression.
Example 11
A FOLR1 Expression Score of .gtoreq.2 Heterogeneous by IHC is a
Patient Selection Criterion for Treatment with IMGN853
[0211] The levels of FOLR1-expression in tumor cell lines were
determined using the an antibody-PE conjugate (FR1-24-PE) and the
QuantiBRITE system. Three ovarian carcinoma cell lines (Igrov-1,
Skov-3 and Ovcar-3), a choriocarcinoma cell line Jeg-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 saturating
concentration (concentration, at which all available binding sites
are occupied by the conjugate). To determine such concentration for
the FR1-24-PE conjugate, we performed binding experiments on a
panel of FOLR1-positive cell lines with various FOLR1 expression.
The cells were incubated with a wide concentration range of
FR1-24-PE conjugate for two hours on ice, washed with FACS buffer
(PBS with 1% BSA), fixed with 1% formaldehyde in PBS and analyzed
on a FACSCalibur flow cytometer. At a concentration of
1.times.10.sup.-8 M the conjugate saturated cell surface binding
sites on all tested cell lines Igrov-1, Jeg-3, Skov-3, Ovcar-3, and
KB. In subsequent binding ABC-experiments FR1-24-PE conjugate was
used at concentration of 1.times.10.sup.-8 M. Each sample was
analyzed in triplicates; several independent experiments were
performed on each cell line. The highest expression was found on KB
cells with the approximate ABC value of 4,000,000.+-.300,000,
followed by Igrov-1 and Jeg-3 cell lines with the ABC values of
400,000.+-.85,000 and 150,000.+-.75,000, respectively. Two cell
lines, Skov-3 and Ovcar-3, had low FOLR1 expression,
20,000.+-.10,000 and 7,000.+-.4,000 ABC, respectively. A
significant experiment-to-experiment variation of ABC values was
observed for Jeg-3 cells, where the ABC-values varied from 40,000
to 300,000. This variability likely reflected some biological
properties of the cell line rather than the assay variability,
since ABC values obtained for the other analyzed cell lines were
much less variable (see table below).
TABLE-US-00016 Experiment-to-experiment variation Cell ABC The
highest ABC The lowest ABC line (Mean .+-. SD, n).sup.i) registered
registered KB 4,000,000 .+-. 300,000, 4 4,500,000 3,800,000 Igrov-1
400,000 .+-. 85,000, 5 480,000 280,000 Jeg-3 150,000 .+-. 75,000,
14 260,000 40,000 Skov-3 20,000 .+-. 10,000 28,000 10,000 Ovcar-3
7,000 .+-. 4,000 10,000 4,000 .sup.i)SD--Standard deviation;
n--number of independent experiments ABC values were determined by
a FACS based assay with FR1-24 PE-labeled antibody and QuantiBRITE
system. Mean .+-. Standard Deviation (SD) was calculated for
independent experiments.
[0212] Potency and specificity of IMGN853 was analyzed against
FOLR1-positive cell lines with a wide range of FOLR1 expression
(the ABC values of the cell lines are provided above). In addition,
FOLR1-negative cell lines Namalwa and SW2 were included in the
experiments. IMGN853 was highly cytotoxic against cells with high
FOLR1 expression KB (4,000,000.+-.300,000 ABC), Igrov-1
(400,000.+-.85,000 ABC) and Jeg-3 (150,000.+-.75,000 ABC), with the
IC.sub.50 values of 0.10.+-.0.01 nM, 0.50.+-.0.07 nM and
1.00.+-.0.05 nM, respectively. The cell-killing activity against
all three cell lines was FOLR1-dependent, since an excess of
unmodified huMov19 (M9346A) antibody (0.5 .mu.M) markedly decreased
potency of the conjugate to the typical non-specific levels (from
10 to 20-fold). IMGN853 was only marginally active against the low
FOLR1 expressors Skov-3 and Ovcar-3 cells (20,000.+-.10,000 and
7,000.+-.4,000 ABC, respectively), and against FOLR1-negative cells
Namalwa and SW2, with the IC.sub.50 values greater than 2 nM. The
cytotoxic activity of IMGN853 against these cell lines was low and
not FOLR1-dependent, as blocking with huMov19 (M9346A) did not
affect it. See FIGS. 19 and 20.
[0213] FFPE samples prepared from mouse xenograft tumor models were
evaluated for FOLR1 positivity using the optimized 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 following cell lines showed the
following staining patterns: Igrov-1, KB, and NCI-H2110 showed
homogeneous 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 intensity; OV-90 showed heterogenous patterns with level 2
intensity; and SKOV3 was negative. Representative photos of tumor
xenografts are provided in FIGS. 21 and 22.
TABLE-US-00017 Parental Cell Line or Tumor Disease Staining
Fragment Indication Final Score Category IGROV-1 Ovarian cancer 1-3
homo 3 homo 1-3 homo 1-3 homo Ishikawa Endometrium 2-3 hetero 3
hetero cancer 1-2 hetero/3 focal 2 hetero/3 focal 2 hetero/3 focal
KB Cervical 3 homo 3 homo cancer 3 homo LXFA737 NSCLC 2 homo 2 homo
2 homo NCI- NSCLC 2-3 homo 3 homo H2110 2 homo OV-90 Ovarian cancer
1-2 hetero 2 hetero Negative.sup.a OVCAR3 Ovarian cancer 1-3 hetero
3 hetero 1-3 hetero SKOV-3 Ovarian cancer Negative Negative
Negative
[0214] A staining threshold (.gtoreq.2 heterogeneous) requires both
a minimal level of expression (staining intensity) and minimum
distribution of staining (percentage of tumor cells expressing
FOLR1). Pre-clinical data provides justification for this threshold
in ovarian carcinoma. Mouse xenograft tumor samples with IHC scores
of .gtoreq.2 heterogeneous exhibit sensitivity to IMGN 853 in vivo.
FFPE samples prepared from mouse xenograft ovarian tumor models
were evaluated for FOLR1 positivity using the optimized and
validated assay described above. Two ovarian carcinoma xenograft
models OVCAR-3, and IGROV-1 showed a heterogeneous or homogeneous
staining pattern with level 3 intensity. The xenograft model
derived from OV-90 ovarian carcinoma cells showed a heterogeneous
staining pattern with level 2 intensity; the Skov-3 ovarian
carcinoma model was FOLR1-negative. IMGN853 was highly active in
the two ovarian models with level 3 FOLR1 intensity and active in
the OV-90 model with level 2 FOLR1 intensity. No activity was
observed in the SKOV-3 model. Xenograft models were also evaluated
for other diseases indications including lung, endometrium, and
cervical tumors and, although correlations were detected between
activity and FOLR1 staining scores, additional samples must be
tested.
[0215] The sensitivity of ovarian tumor xenograft models to IMGN853
versus the level of FOLR1 expression
TABLE-US-00018 In vivo activity (5 mg/kg Intensity score, Xenograft
of IMGN853, single dose) distribution OVCAR3 Highly active 3
heterogeneous IGROV-1 Highly active 3 homogeneous OV-90 Active 2
heterogeneous SKOV-3 Inactive Negative
The sensitivity of other tumor xenograft models to IMGN853 versus
the level of FOLR1 expression
TABLE-US-00019 Type of In vivo activity (5 mg/kg Intensity score,
Xenograft tumor of IMGN853, single dose) distribution NCI- NSCLC
Highly active 3 homogeneous H2110 Ishikawa Endometrium Inactive 2
homogeneous KB Cervical Highly active 3 homogeneous
[0216] All publications, patents, patent applications, internet
sites, and accession numbers/database sequences (including both
polynucleotide and polypeptide sequences) cited herein are hereby
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication, patent, patent
application, internet site, or accession number/database sequence
were specifically and individually indicated to be so incorporated
by reference.
TABLE-US-00020 SEQUENCES human folate receptor 1 SEQ ID NO: 1
MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNA
KHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSY
LYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQV
DQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGW
NWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVS
NYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWA AWPFLLSLALMLLWLLS human
folate receptor 1 nucleic acid sequence SEQ ID NO: 2
atggctcagcggatgacaacacagctgctgctccttctag
tgtgggtggctgtagtaggggaggctcagacaaggattgc
atgggccaggactgagcttctcaatgtctgcatgaacgcca
agcaccacaaggaaaagccaggccccgaggacaagttgcat
gagcagtgtcgaccctggaggaagaatgcctgctgttctac
caacaccagccaggaagcccataaggatgtttcctacctat
atagattcaactggaaccactgtggagagatggcacctgcc
tgcaaacggcatttcatccaggacacctgcctctacgagtg
ctcccccaacttggggccctggatccagcaggtggatcaga
gctggcgcaaagagcgggtactgaacgtgcccctgtgcaaa
gaggactgtgagcaatggtgggaagattgtcgcacctccta
cacctgcaagagcaactggcacaagggctggaactggactt
cagggtttaacaagtgcgcagtgggagctgcctgccaacct
ttccatttctacttccccacacccactgttctgtgcaatga
aatctggactcactcctacaaggtcagcaactacagccgag
ggagtggccgctgcatccagatgtggttcgacccagccca
gggcaaccccaatgaggaggtggcgaggttctatgctgca
gccatgagtggggctgggccctgggcagcctggcctttcc
tgcttagcctggccctaatgctgctgtggctgctcagc huMov19 vHC SEQ ID NO: 3
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQ
SPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNT
AHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSS huMov19 vLCv1.00 SEQ ID
NO: 4 DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHW
YHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLN
ISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR huMov19 vLCv1.60 SEQ ID NO: 5
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHW
YHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLT
ISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR
Sequence CWU 1
1
111257PRTHomo sapiens 1Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu
Leu Leu Val Trp Val1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile
Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys
His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln
Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser
Gln Glu Ala His Lys Asp Val Ser Tyr65 70 75 80Leu Tyr Arg Phe Asn
Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe
Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly
Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120
125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu
130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys
Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val
Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro
Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val
Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp
Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg
Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235
240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu
245 250 255Ser2771DNAHomo sapiens 2atggctcagc ggatgacaac acagctgctg
ctccttctag tgtgggtggc tgtagtaggg 60gaggctcaga caaggattgc atgggccagg
actgagcttc tcaatgtctg catgaacgcc 120aagcaccaca aggaaaagcc
aggccccgag gacaagttgc atgagcagtg tcgaccctgg 180aggaagaatg
cctgctgttc taccaacacc agccaggaag cccataagga tgtttcctac
240ctatatagat tcaactggaa ccactgtgga gagatggcac ctgcctgcaa
acggcatttc 300atccaggaca cctgcctcta cgagtgctcc cccaacttgg
ggccctggat ccagcaggtg 360gatcagagct ggcgcaaaga gcgggtactg
aacgtgcccc tgtgcaaaga ggactgtgag 420caatggtggg aagattgtcg
cacctcctac acctgcaaga gcaactggca caagggctgg 480aactggactt
cagggtttaa caagtgcgca gtgggagctg cctgccaacc tttccatttc
540tacttcccca cacccactgt tctgtgcaat gaaatctgga ctcactccta
caaggtcagc 600aactacagcc gagggagtgg ccgctgcatc cagatgtggt
tcgacccagc ccagggcaac 660cccaatgagg aggtggcgag gttctatgct
gcagccatga gtggggctgg gccctgggca 720gcctggcctt tcctgcttag
cctggcccta atgctgctgt ggctgctcag c 7713118PRTArtificial
SequencehuMov19 vHC 3Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Gly Tyr 20 25 30Phe Met Asn Trp Val Lys Gln Ser Pro
Gly Gln Ser Leu Glu Trp Ile 35 40 45Gly Arg Ile His Pro Tyr Asp Gly
Asp Thr Phe Tyr Asn Gln Lys Phe 50 55 60Gln Gly Lys Ala Thr Leu Thr
Val Asp Lys Ser Ser Asn Thr Ala His65 70 75 80Met Glu Leu Leu Ser
Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys 85 90 95Thr Arg Tyr Asp
Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val
Thr Val Ser Ser 1154112PRTArtificial SequencehuMov19 vLCv1.00 4Asp
Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly1 5 10
15Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala
20 25 30Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln
Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val
Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu
Asn Ile Ser65 70 75 80Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Ser Arg 85 90 95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 1105112PRTArtificial
SequencehuMov19 vLCv1.60 5Asp Ile Val Leu Thr Gln Ser Pro Leu Ser
Leu Ala Val Ser Leu Gly1 5 10 15Gln Pro Ala Ile Ile Ser Cys Lys Ala
Ser Gln Ser Val Ser Phe Ala 20 25 30Gly Thr Ser Leu Met His Trp Tyr
His Gln Lys Pro Gly Gln Gln Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala
Ser Asn Leu Glu Ala Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly
Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Pro Val Glu Ala
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95Glu Tyr Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
11065PRTArtificial sequencehuMov19 vHC CDR1 6Gly Tyr Phe Met Asn1
5717PRTArtificial sequenceKabat Defined Mov19 HC CDR2 Human 7Arg
Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe Gln1 5 10
15Gly89PRTArtificial sequencehuMov19 vHC CDR3 8Tyr Asp Gly Ser Arg
Ala Met Asp Tyr1 5915PRTArtificial sequencehuMov19 vLC CDR1 9Lys
Ala Ser Gln Ser Val Ser Phe Ala Gly Thr Ser Leu Met His1 5 10
15107PRTArtificial sequencehuMov19 vLC CDR2 10Arg Ala Ser Asn Leu
Glu Ala1 5119PRTArtificial sequencehuMov19 vLC CDR3 11Gln Gln Ser
Arg Glu Tyr Pro Tyr Thr1 5
* * * * *