NZ756675A - Antibodies and assays for detection of folate receptor 1 - Google Patents
Antibodies and assays for detection of folate receptor 1Info
- Publication number
- NZ756675A NZ756675A NZ756675A NZ75667514A NZ756675A NZ 756675 A NZ756675 A NZ 756675A NZ 756675 A NZ756675 A NZ 756675A NZ 75667514 A NZ75667514 A NZ 75667514A NZ 756675 A NZ756675 A NZ 756675A
- Authority
- NZ
- New Zealand
- Prior art keywords
- folrl
- antibody
- cancer
- seq
- antigen
- Prior art date
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Abstract
The invention generally relates to nucleic acids and vectors comprising nucleic acids which encode for antibodies that bind to human folate receptor and diagnostic assays for folate receptor 1-based therapies. Said antibodies bind to an epitope of folate receptor 1 (FOLR1) which comprises at least one, two or three N-glycosylated amino acids. Methods of using the antibodies to monitor therapy are further provided.
Description
DIES AND ASSAYS FOR ION OF
FOLATE RECEPTOR 1
FIELD OF THE INVENTION
This application is a divisional of New d Patent Application No. 717140, filed
on 29 August 2014, and claims the t of US Provisional Application Nos. 61/872,407 filed
on 30 August 2013, 61/875,475 filed on 9 September 2013, and 61/940,184 filed on 14 February
2014, each of which is incorporated herein by reference in its entirety.
[0001a] The field of this invention generally relates to diagnostic assays and kits for folate
receptor 1-based therapies and dies that bind to human folate receptor 1.
BACKGROUND OF THE INVENTION
Cancer is one of the leading causes of death in the developed world, with over one
million people diagnosed with cancer and 500,000 deaths per year in the United States alone.
Overall it is estimated that more than 1 in 3 people will develop some form of cancer during their
me. There are more than 200 ent types of cancer, four of which— breast, lung,
colorectal, and prostate— account for over half of all new cases (Jemal et al., 2003, Cancer J.
Clin. 53:5-26).
Folate 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.
FOLR1 is overexpressed in the vast majority of ovarian cancers, as well as in many
uterine, endometrial, pancreatic, renal, lung, and breast cancers, while the sion of FOLR1
on normal tissues is restricted to the apical membrane of epithelial cells in the kidney proximal
tubules, alveolar pneumocytes of the lung, r, testes, choroid plexus, and thyroid (Weitman
SD, et al, Cancer Res 52: 3396-3401 (1992); Antony AC, Annu Rev Nutr 16: 501-521 (1996);
Kalli KR, et al. Gynecol Oncol 108: 619-626 (2008)). This expression pattern of FOLR1 makes
it a desirable target for FOLR1 -directed cancer therapy.
Because ovarian cancer is typically asymptomatic until advanced stage, it is often
diagnosed at a late stage and has poor sis when treated with tly available ures,
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 VN et al,
Obstet Gynecol Surv 64: 548-560 (2009)). Thus there is a clear unmet medical need for more
effective diagnostics for ovarian cancers.
Some previous assays used to detect shed FOLRl are not sufficiently specific
to FOLRl. For example, some assays do not guish between FOLRl and other folate
receptor family members (FOLR2, 3, & 4) or report values for total FBP (Folate Binding
Protein). Additionally, some assays require that human samples (e.g., plasma) be pre-treated
with a light acid wash step to dissociate folic acid from the receptor. Some assay results may
also have inaccuracies due to competitive effects between the antibody therapy and
stic dy. Additionally, many commercially available kits are traditionally
unreliable both in their reagents, and in their lot-to-lot stability. Evaluations of these kits
have given very mixed results, and are ed for ch use only. Many e that the
human sample be pre-diluted before is to reduce the chance of false positives due to the
"matrix effect." Thus, there is a clear need for highly sensitive and accurate diagnostic assays
that can detect a clinically relevant dynamic range of FOLRl as a companion for FOLRl-
based therapies.
SUMMARY OF THE INVENTION
Anti-FOLRl antibodies and antigen—binding fragments thereof as well as
methods for detecting FOLRl, sing FOLRl—mediated diseases and disorders (such as
), monitoring the efficacy of anti-FOLRl therapies, optimizing anti-FOLRl therapies,
and stratifying patients are all provided herein.
The anti-FOLRl antibodies provided herein can have a diagnostic role. For
example, the anti-FOLRl antibodies provided herein to distinguish n tumor and non-
tumor cells or tissues or to identify tumor types, subtypes, or grades. In one embodiment, an
anti-FOLRl antibody ed herein and/or a FOLRl-detection assay provided herein can
be used to distinguish between es of non-small cell lung cancer (NSCLC) including
adenocarcinoma and squamous cell carcinoma as described herein. In another embodiment,
an anti-FOLRl antibody provided herein and/or a FOLRl—detection assay provided herein
can be used to rule out a type of cancer (e.g., to determine that a cell or tissue is not a type of
cancer), for example, sarcoma.
In some embodiments, an antibody or antigen-binding nt thereof
provided herein can specifically bind to an epitope of FOLRl wherein the epitope comprises
at least one, at least two, or three N—glycoslated amino acids. Glycosylation can be al
for membrane localization. See e.g., Yan et al., J. Am. Soc. Nephol. 13: 1385-1389 (2002).
Advantageously, the antibodies and antigen—binding fragments herein can detect FOLRl
expression on cell membranes and detect a clinically relevant dynamic range of FOLRl. The
more discreet staining obtained with the antibodies and antigen-binding fragments ed
herein allows for discrimination among samples all grouped together as high expression
levels (with a score of 3) using antibodies that bind to different FOLRl epitopes, lack
sufficient specificity, and/or lack sufficient sensitivity.
In some embodiments, an antibody or antigen-binding fragment f
ed herein can specifically bind to the same FOLR] epitope as an antibody selected
from the group consisting of: (a) an antibody comprising the polypeptide of SEQ ID NO:27
and the polypeptide of SEQ ID NO:28; (b) an dy comprising the polypeptide of SEQ
ID N029 and the polypeptide of SEQ ID NO:30; (c) an antibody comprising the polypeptide
of SEQ ID NO:3l and the polypeptide of SEQ ID NO:32; (d) an antibody comprising the
polypeptide of SEQ ID NO:62 and the polypeptide of SEQ ID NO:63 or SEQ ID NO:64; and
(e) an antibody comprising the polypeptide of SEQ ID NO:65 and the ptide of SEQ ID
NO:66 or SEQ ID NO:67. In some embodiments, the epitope comprises an N-glycosylated
amino acid.
In some embodiments, an antibody or antigen-binding fragment thereof
provided herein can specifically bind to FOLRl, wherein said antibody or fragment thereof
competitively inhibits binding to FOLRl of an antibody selected from the group consisting
of: (a) an dy comprising the polypeptide of SEQ ID NO:27 and the polypeptide of SEQ
ID NO:28; (b) an dy comprising the polypeptide of SEQ ID N029 and the polypeptide
of SEQ ID NO:30; (c) an antibody comprising the polypeptide of SEQ ID N031 and the
ptide of SEQ ID NO:32; (d) an antibody comprising the polypeptide of SEQ ID NO:62
and the polypeptide of SEQ ID NO:63 or SEQ ID NO:64; and (e) an antibody comprising the
polypeptide of SEQ ID NO:65 and the polypeptide of SEQ ID NO:66 or SEQ ID NO:67.
In some embodiments, the antibody or antigen-binding fragment thereof
comprises the VH CDRl-3 and VL CDR1—3 polypeptide sequences ed from the group
consisting of: (a) SEQ ID NOS23-8, respectively; (b) SEQ ID 14, respectively; (c)
SEQ ID NOS215-20, respectively; (d) SEQ ID NOS:21-26, respectively; (e) SEQ ID NOS: 3-5
and SEQ ID NOS: 59, 7, and 8, tively; (t) SEQ ID NOS: 3, 60, and 5 and SEQ ID NOS:
6-8, respectively; (g) SEQ ID NOS: 3, 61, and 5 and SEQ ID NOS: 6-8, respectively; (h) SEQ
ID NOS: 3, 60, and 5 and SEQ ID NOS: 59, 7, and 8, respectively; and (i) SEQ ID NOs: 3, 61,
and 5 and SEQ ID NOS: 59, 7, and 8, tively.
In some ments, an antibody or antigen-binding fragment thereof
provided herein can specifically bind to FOLRl, wherein the antibody or fragment thereof
comprises the VH CDRl-3 and VL CDRl—3 polypeptide sequences selected from the group
consisting of: (a) SEQ ID NOs23-8, respectively; (b) SEQ ID NOs:9-l4, respectively; (c)
SEQ ID NOs:lS-20, respectively; (d) SEQ ID -26, tively; (e) SEQ ID NOs: 3-5
and SEQ ID NOs: 59, 7, and 8, respectively; (f) SEQ ID NOs: 3, 60, and 5 and SEQ ID NOs:
6-8, respectively; (g) SEQ ID NOs: 3, 61, and 5 and SEQ ID NOs: 6-8, respectively; (h) SEQ
ID NOs: 3, 60, and 5 and SEQ ID NOs: 59, 7, and 8, respectively; (i) SEQ ID NOs: 3, 61, and
and SEQ ID NOs: 59, 7, and 8, respectively; and (i) variants of (a) to (i) comprising 1, 2, 3,
or 4 conservative amino acid substitutions.
In some ments, the antibody or antigen-binding fragment thereof
comprises polypeptide sequences that are at least 90%, at least 95%, or at lesast 99%
identical to polypeptide sequences selected from the group consisting of: (a) SEQ ID NO:27
and SEQ ID NO:28; (b) SEQ ID N029 and SEQ ID NO:30; (c) SEQ ID NO:3l and SEQ ID
NO:32; (d) SEQ ID NO:62 and SEQ ID NO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and
SEQ ID NO:66 or SEQ ID NO:67; (f) SEQ ID NO:68 and SEQ ID NO:69. In some
embodiments, the polypeptide sequences comprise, consist essentially of, or consist of the
amino acids of sequences selected from the group consisting of: (a) SEQ ID NO:27 and SEQ
ID NO:28; (b) SEQ ID N029 and SEQ ID NO:30; (c) SEQ ID N031 and SEQ ID NO:32;
(d) SEQ ID NO:62 and SEQ ID NO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID
NO:66 or SEQ ID NO:67; (f) SEQ ID NO:68 and SEQ ID NO:69.
In some embodiments, an antibody or antigen-binding fragment thereof
provided herein can specifically bind to FOLRI, wherein the antibody or fragment f
comprises a humanized heavy chain variable region comprising CDRl, CDR2, and CDR3
regions comprising the amino acids of SEQ ID NO:51, SEQ ID NO:52 or 53, and SEQ ID
NO:54, respectively, a humanized light chain variable region comprising CDRl, CDR2, and
CDR3 regions comprising the amino acids of SEQ ID NO:48, SEQ ID NO:49, and SEQ ID
N0250, respectively, and a murine constant region. In some embodiments, the humanized
heavy chain variable region comprises the amino acids of SEQ ID NO:45 and the humanized
light chain variable region comprises the amino acids of SEQ ID NO:47.
In some embodiments, the antibody or antigen-binding nt thereof is
recombinantly produced. In some embodiments, the antibody or antigen-binding fragment
thereof is murine, non-human, humanized, ic, resurfaced, or human. In some
embodiments, the antibody or antigen—binding fragment thereof binds to human FOLRl but
not FOLR2 or FOLR3. In some embodiments, the dy or n-binding fragment
f is a full length antibody. In some embodiments, the antibody or antigen-binding
fragment thereof is an antigen-binding fragment. In some embodiments, the dy or
antigen-binding fragment f ses, consists essentiall of, or consist of a Fab, Fab',
F(ab')2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody,
IgGACHZ, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain dy, DVD-
Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.
In some embodiments, a polypeptide provided herein can specifically bind
FOLRl, wherein the polypeptide comprises sequences selected from the group consisting of:
(a) SEQ ID NOsz3-8, respectively; (b) SEQ ID 14, respectively; (0) SEQ ID NOs:15-
, respectively; (d) SEQ ID -26, respectively; (e) SEQ ID NOs: 3-5 and SEQ ID
NOs: 59, 7, and 8, respectively; (f) SEQ ID NOS: 3, 60, and 5 and SEQ ID NOs: 6-8,
respectively; (g) SEQ ID NOS: 3, 61, and 5 and SEQ ID NOS: 6-8, respectively (h) SEQ ID
NOs: 3, 60, and 5 and SEQ ID NOs: 59, 7, and 8, respectively; (i) SEQ ID NOs: 3, 61, and 5
and SEQ ID NOs: 59, 7, and 8, tively; and (j) variants of (a) to (i) comprising 1, 2, 3, or
4 conservative amino acid substitutions. In some embodiments, the polypeptide comprises
sequences that are at least 90%, at least 95%, or at least 99% identical to sequences selected
from the group consisting of: (a) SEQ ID N027 and SEQ ID NO:28; (b) SEQ ID N029 and
SEQ ID NO:30; (c) SEQ ID NO:31 and SEQ ID NO:32; (d) SEQ ID NO:62 and SEQ ID
NO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ ID NO:67; and (f)
SEQ ID NO:68 and SEQ ID NO:69. In some embodiments, the polypeptide comprises the
amino acids of (a) SEQ ID N027 and SEQ ID NO:28; (b) SEQ ID N029 and SEQ ID
NO:30; (c) SEQ ID N031 and SEQ ID NO:32; (d) SEQ ID NO:62 and SEQ ID NO:63 or
SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ ID NO:67; or (f) SEQ ID
NO:68 and SEQ ID NO:69.
In some embodiments, the antibody, antigen—binding fragment thereof, or
polypeptide binds to FOLRl with a Kd of about 0.5 to about 10 nM. In some embodiments,
the antibody, antigen-binding fragment f, or polypeptide binds to a human FOLRl with
a Kd of about 1.0 11M or better. In some embodiments, the binding affinity is measured by
flow cytometry, Biacore, ELISA, or radioimmunoassay.
In some embodiments, the antibody, antigen-binding nt thereof, or
polypeptide binds to an epitope of FOLRl comprising an amino acid that is N—glycosylated.
In some embodiments, the antibody, antigen-binding fragment thereof, or
polypeptide is detectably labeled.
In some embodiments, a cell provided herein produces the antibody, antigen-
binding fragment thereof, or ptide. In some embodiments, the cell is isolated.
Methods of making the dy, antigen-binding fragment thereof, or
polypeptide are also provided. The methods can comprise (a) culturing a cell provided
herein; and (b) ing the antibody, antigen—binding fragment f, or polypeptide from
the cultured cell.
Compositions comprising the antibody, antigen-binding fragment thereof, or
polypeptide are also ed. In some ments, the composition comprises the the
antibody, antigen-binding fragment thereof, or polypeptide and buffer selected from the
group consisting of: a FACS buffer, an IHC buffer, and an ELISA buffer.
Methods of using the antibody, antigen-binding fragment thereof, or
ptide are also provided.
In some embodiments, a method of detecting FOLRl expression in a sample
comprises contacting the sample with an antibody, antigen-binding fragment thereof,
polypeptide, or composition ed herein. In some embodiments, the antibody or antigen-
binding nt thereof is detectably labeled. In some embodiments, the label is selected
from the group ting of immunofluorescent label, chemiluminescent label,
phosphorescent label, enzyme label, radiolabel, avidin/biotin, colloidal gold particles, colored
particles and magnetic particles. In some embodiments, the FOLRl expression is determined
by radioimmunoassay, Western blot assay, cytometry, immunofluorescent assay, enzyme
immunoassay, immunoprecipitation assay, chemiluminescent assay, or immunohistochemical
assay. In some embodiments, the cytometry is flow cytometry. In some embodiments, the
FOLRl expression is determined by IHC.
In some embodiments, a method for increasing the efficacy of cancer y
with an active agent comprising an OLRl antibody or antigen-binding fragment
thereof, comprises stering the active agent to a subject having cancer, wherein
increased expression of FOLRI has been detected in a cancerous sample from the subject
using an antibody, antigen-binding fragment thereof, polypeptide or composition provided
herein.
In some embodiments, a method for identifying a cancer likely to respond to
an active agent comprising an anti-FOLR] antibody or antigen-binding fragment thereof
comprises: (a) contacting a biological sample comprising cells from the cancer with the
antibody, antigen-binding fragment thereof, ptide or composition provided herein; (b)
detecting binding of the antibody, antibody—fragment, or polypeptide to FOLRl in the
biological sample of (a); (c) assigning a score to the binding of step (b), wherein the score is
assigned based on comparison to one or more nce samples; and (d) comparing the score
in step (c) to the score of a reference tissue or cell, n a score for the cancer FOLRl
level that is greater than the score for a normal or low FOLRl expressing reference sample or
a score for the cancer FOLRl level that is equal to or greater than the score for a high FOLRl
sing reference sample fies the cancer as likely to respond to an anti-FOLRl
antibody.
In some ments, a method of treating a patient having cancer
ses:(a) determining a FOLRI expression score from a detection of FOLRl expression
in a cancerous sample obtained from the patient, wherein the detection is performed using an
antibody, antigen-binding fragment thereof, polypeptide or composition provided herein; and
(b) administering an active agent comprising an anti-FOLR] antibody or antigen-binding
fragment thereof to the patient if the score indicates the t will benefit from
administration of the active agent.
In some embodiments, a method of treating a patient having cancer comprises
(a) determining a FOLRl expression score from a detection of FOLRl expression in a
cancerous sample ed from the patient, wherein the detection is performed using an
antibody, antigen-binding fragment thereof, polypeptide or composition provided herein; and
(b) instructing a healthcare er to administer an active agent comprising an anti-FOLRl
antibody or n-binding fragment thereof to the patient if the score indicates the patient
will benefit from administration of the active agent.
In some embodiments, a method of treating a patient having cancer comprises:
(a) ting a cancerous sample taken from a patient having cancer for determining a
FOLRl expression score from a detection of FOLRl expression using an antibody, antigen-
binding fragment thereof, polypeptide, or composition provided herein; and (b) administering
an active agent comprising an anti-FOLR] antibody or antigen-binding nt thereof to
the patient if the score indicates the patient will benefit from administration of the active
agent.
In some embodiments, a method of treating a patient having cancer comprises:
(a) detecting FOLRl expression in a ous sample obtained from the patient, wherein the
detection is med using an antibody, antigen-binding fragment thereof, polypeptide, or
composition provided herein; (b) determining a FOLRl expression score for the cancerous
sample; and (c) administering an active agent comprising an anti-FOLRl dy or antigenbinding
fragment thereof to the patient if the score indicates the patient will benefit from
administration of the active agent.
In some embodiments, a method of treating a patient having cancer comprises:
(a) administering to the patient a fixed dose of an active agent comprising an anti-FOLRl
antibody or antigen-binding fragment thereof; (b) detecting the patient's FOLRl relative to
the FOLRl level in a reference sample, wherein the detection is performed using an antibody,
antigen-binding fragment thereof, polypeptide, or composition provided herein; and (c)
increasing the amount or frequency of uent fixed doses if the patient’s FOLRl level is
elevated.
In some embodiments, a method of optimizing a therapeutic regimen with an
active agent comprising an anti-FOLRl antibody or n-binding fragment thereof for a
subject having cancer comprises: (a) administering an increased dose of an active agent
comprising an anti—FOLRl antibody or n-binding fragment thereof to a subject having
cancer wherein an sed expression of FOLRl in the subject has been detected using an
antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; or
(b) administering a decreased dose of the active agent to a subject having cancer wherein a
decreased expression of FOLRl in the subject has been ed.
In some embodiments, a method of optimizing a therapeutic regimen with an
active agent comprising an anti-FOLRl dy or antigen-binding fragment f for a
subject having cancer comprises: (a) detecting the level of FOLRl sion in a ous
sample from the subject using an antibody, n-binding fragment thereof, polypeptide, or
composition provided herein; (b) determining a FOLRl expression score for the cancerous
sample; and (c) administering an increased dose of an active agent comprising an anti-
FOLRl antibody or antigen-binding fragment thereof to the subject if the score is low or
administering a decreased dose of the active agent to the subject if the score is high.
In some embodiments, a method of decreasing FOLRl-expressing cancer cells
in a cancer patient comprises: (a) detecting the FOLRl level in a cancerous sample taken
from a patient, ed to the FOLRI level in a nce sample using an antibody,
antigen-binding fragment f, polypeptide, or composition provided herein; and (b)
administering to the patient a fixed dose of an active agent comprising an anti-FOLRl
dy or antigen-binding fragment thereof if the t’s FOLRl level is elevated
compared to the reference sample; wherein the administration of the active agent decreases
the number of FOLRl-expressing cancer cells in the patient. In some embodiments, a
method of treating cancer in a patient comprises: (a) detecting the FOLRl level in a
cancerous sample taken from a patient, compared to the FOLRl level in a reference sample
using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided
herein; and (b) administering to the patient a fixed dose of an active agent comprising an anti-
FOLRl antibody or antigen-binding fragment f if the t’s FOLRl level is elevated
compared to the reference sample; n the administration of the active agent decreases
the size of a FOLRl-expressing tumor or decreases CA125 levels.
In some embodiments, a method of decreasing FOLRl-expressing cancer cells
in a cancer t comprises:(a) administering to a t having a cancer a fixed dose of an
active agent comprising an anti-FOLR] antibody or antigen-binding fragment thereof; (b)
detecting the patient's FOLRl level relative to the FOLRl level in a reference sample using
an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein;
and (0) increasing the amount or frequency of subsequent fixed doses if the patient’s FOLRl
level is ed ed to the reference sample; wherein the administration of the active
agent decreases the number of FOLRl—expressing cancer cells in the patient. In some
embodiments, a method of treating cancer in a patient comprises: (a) administering to a
patient having a cancer a fixed dose of an active agent comprising an anti-FOLRl antibody or
antigen-binding fragment thereof; (b) detecting the patient's FOLRl level ve to the
FOLRl level in a reference sample using an antibody, antigen-binding fragment thereof,
polypeptide, or composition provided herein; and (0) increasing the amount or frequency of
subsequent fixed doses if the patient’s FOLRl level is elevated compared to the reference
sample; n the administration of the active agent decreases the size of a FOLRl-
expressing tumor or decreases CAl25 levels.
In some embodiments, a method of monitoring therapeutic efficacy of a fixed
dose of an active agent comprising an anti-FOLRl antibody or antigen-binding fragment
thereof in a patient comprises: (a) detecting a first FOLRl level in a biological sample from a
patient having cancer using an antibody, n—binding fragment thereof, polypeptide, or
composition provided herein; (b) administering to the patient a fixed dose of an active agent
comprising an anti-FOLRI antibody or antigen-binding fragment; (c) detecting a second
FOLRl level in a biological sample from the patient following active agent administration,
wherein the detecting is med using an antibody, n-binding fragment f,
polypeptide, or composition provided ; and (d) comparing the second FOLRl level to
the first FOLRl level; n a decrease between the first and second FOLRl level
indicates therapeutic efficacy.
In some embodiments, a method of identifying a subject having a cancer as
likely to respond to a low dose anti-FOLRl treatment regimen, comprises: (a) contacting a
biological sample comprising cells from the cancer with an antibody, antigen-binding
fragment thereof, polypeptide, or composition provided herein; (b) detecting binding of the
antibody, antigen-binding fragment, or polypeptide to the ical sample of (a); (c)
ing a score to the binding of step (b), wherein the score is assigned based on
comparison to one or more nce samples; and (d) comparing the score in step (c) to the
score of a reference tissue or cell, wherein a score for the cancer FOLRl level that is greater
than the score for a normal or low FOLR] sing reference sample or a score for the
cancer FOLRl level that is equal to or greater than the score for a high FOLRl expressing
reference sample identifies the cancer as likely to respond to a low dose OLRl
treatment.
In some embodiments, a method of identifying a cancer as sensitive to
treatment with an anti—FOLRI active agent, comprises: (a) detecting the level of FOLRl
expression in a cancerous sample from the cancer using an antibody, antigen-binding
fragment thereof, polypeptide, or composition provided , wherein the detecting
ses the use of a method that distinguishes between ng intensity or staining
uniformity in a FOLRl expressing ous sample as compared to staining intensity or
staining mity in one or more reference samples; (b) determining a FOLRl staining
intensity or staining uniformity score for the cancerous sample; and (c) comparing the
FOLRl staining intensity or staining uniformity score determined in step (b) to a relative
value determined by measuring FOLRl protein expression in at least one reference sample,
wherein the at least one reference sample is a tissue, cell, or cell pellet sample which is not
sensitive to treatment with an active agent comprising an anti-FOLRl antibody or antigenbinding
fragment thereof and wherein a FOLRl staining intensity score for the cancerous
sample determined in step (b) that is higher than the relative value identifies the cancer as
being sensitive to treatment with the active agent.
In some embodiments, a method of identifying a cancer as sensitive to
treatment with an anti-FOLRl active agent, comprises: (a) detecting the level of FOLRl
expression in a cancerous sample from the cancer using an dy, n-binding
fragment thereof, polypeptide, or composition provided herein, wherein the detecting
comprises the use of a method that specifically stains ne FOLRl in a FOLRl
expressing cancerous sample as compared to membrane FOLRl in one or more reference
samples; (b) determining a FOLRl score for the cancerous sample; and (c) comparing the
FOLRl score determined in step (b) to a relative value determined by measuring FOLRl in at
least one reference sample, wherein the at least one reference sample is a tissue, cell, or cell
pellet sample which is not ive to treatment with an active agent comprising an anti-
FOLRl antibody or antigen-binding fragment thereof and wherein a FOLRl score for the
cancerous sample determined in step (b) that is higher than the relative value identifies the
cancer as being sensitive to treatment with the active agent.
In some embodiments, a method of identifying a cancer as sensitive to
treatment with an anti-FOLRl active agent, comprises: (a) detecting the level of FOLRl
expression in a cancerous sample from the cancer using an antibody, antigen-binding
fragment thereof, ptide, or ition provided herein, wherein the detecting
ses the use of a method that distinguishes n staining intensity or ng
uniformity in a FOLRI expressing cancerous sample as compared to staining intensity or
staining uniformity in one or more reference s; (b) determining a FOLRl ng
intensity or staining uniformity score for the cancerous sample; and (c) comparing the
FOLRl staining intensity or staining uniformity score determined in step (b) to a relative
value determined by measuring FOLRl n expression in at least one reference ,
wherein the at least one reference sample is a tissue, cell, or cell pellet sample which is
sensitive to treatment with an active agent comprising an OLRl antibody or antigen-
binding fragment f and wherein a FOLRl staining intensity score for the cancerous
sample determined in step (b) that is greater than or equal to the relative value identifies the
cancer as being sensitive to treatment with the active agent.
In some embodiments, a method of identifying a cancer as ive to
treatment with an anti-FOLRl active agent, comprises: (a) detecting the level of FOLRl
expression in a cancerous sample from the cancer using an antibody, antigen-binding
fragment thereof, polypeptide, or composition provided herein, wherein the detecting
comprises the use of a method that specifically stains membrane FOLRl in a FOLRl
expressing cancerous sample as compared to membrane FOLRl in one or more reference
s; (b) determining a FOLRl score for the cancerous sample; and (c) comparing the
FOLRl score determined in step (b) to a relative value determined by measuring FOLRl in at
least one nce sample, wherein the at least one reference sample is a tissue, cell, or cell
pellet sample which is sensitive to treatment with an active agent comprising an anti-FOLRl
antibody or antigen-binding fragment thereof and wherein a FOLR] score for the cancerous
sample determined in step (b) that is greater than or equal to the relative value identifies the
cancer as being sensitive to treatment with the active agent.
In some embodiments, the method further comprises administering an active
agent comprising an anti-FOLRl antibody or antigen—binding fragment thereof to the subject
from whom the cancerous sample or biological sample was obtained.
In some embodiments, the patient's FOLRl level is detected in a cancerous
sample or biological sample obtained from the patient. In some embodiments, the cancerous
sample or biological sample is a bodily fluid, cell, or tissue sample. In some embodiments,
the cell is a circulating tumor cell. In some ments, the bodily fluid is blood, ascites,
urine, plasma, serum, or peripheral blood.
In some embodiments, the FOLRl is membrane localized FOLRl.
In some embodiments, the FOLRl is shed FOLRl.
In some embodiments, the detecting is by enzyme linked immunosorbent
assay (ELISA).
In some embodiments, the detecting is by immunohistochemistry (IHC). In
some embodiments, the IHC is calibrated IHC that can distinguish different levels of FOLRl
expression. In some embodiments, the IHC produces a range of ng intensity for samples
having low cell surface FOLRl expression, intermediate FOLRl cell e expression, or
high FOLRl cell surface expression. In some embodiments, the IHC distinguishes between
staining intensity and staining uniformity in a FOLRl expressing cancerous sample or
ical sample as compared to a reference sample. In some embodiments, the IHC detects
membrane FOLRl. In some embodiments, the IHC is performed manually. In some
embodiments, the IHC is performed using an automated system.
In some embodiments, a FOLRl score is ined from the IHC.
In some embodiments, a score of at least 1 indicates an increased expression
of FOLRl and identifies the cancer as likely to d to an active agent comprising an anti-
FOLRl antibody or antigen-binding fragment thereof.
In some embodiments, a score of at least 2, at least 2 homo (>75%
uniformity), or at least 2 hetero (25-75% uniformity) identifies the cancer as likely to d
to an active agent comprising an anti—FOLRI dy or n—binding fragment thereof.
In some embodiments, the cancer is lung cancer or endometrial cancer. In some
embodiments, a score of at least 3, at least 3 homo (>75% uniformity), or at least 3 hetero
(25-75% uniformity) identifies the cancer as likely to d to an active agent comprising
an anti-FOLRl antibody or antigen-binding fragment thereof. In some embodiments, the
cancer is lung , endometrial cancer, or ovarian cancer.
In some embodiments, an H—score of at least 50 identifies a cancer as likely to
respond to an active agent comprising an anti—FOLRl antibody or antigen-binding fragment
thereof. In some embodiments, an H-score of at least 75 identifies an ovarian cancer as likely
to respond to an active agent comprising an OLRl antibody or antigen-binding
fragment f. In some embodiments, an H-score of at least 50 identifies an NSCLC as
likely to respond to an active agent sing an anti-FOLRl antibody or antigen-binding
nt thereof. In some embodiments, an H-score of at least 50 identifies an endometrial
cancer as likely to respond to an active agent sing na anti-FOLRl antibody or antigen-
binding fragment thereof. In one embodiment, an H-score is determined using the FOLRl-
21 antibody.
In some ments, at least 25% of FOLR] membrane expression in an
ovarian tumor sample with an ity of at least 3 fies the ovarian cancer as likely to
respond to an active agent comprising an anti-FOLRl antibody or antigen-binding fragment
thereof. In some embodiments, at least 25% of FOLRl membrane expression in an NSCLC
sample with an intensity of at least 2 identifies the NSCLC as likely to respond to an active
agent sing an anti—FOLRl antibody or antigen—binding fragment thereof. In some
embodiments, at least 25% of FOLRl membrane expression in an endometrial tumor sample
with an intensity of at least 2 identifies the endometrial cancer as likely to respond to an
active agent sing an anti-FOLRl antibody or antigen-binding fragment thereof. In one
embodiment, the expression score is determined using the FOLRl-2.l antibody.
In some embodiments, a score of at least 1 indicates an increased expression
of FOLRl and that the patient will benefit from administration of an active agent comprising
an anti-FOLRl antibody or antigen-binding fragment thereof. In some embodiments, a score
of at least 2, at least 2 homo (>75% uniformity), or at least 2 hetero (25-75% uniformity)
indicates that the patient will benefit from administration of an active agent comprising an
anti-FOLRl antibody or antigen-binding fragment thereof. In some embodiments, the cancer
is lung cancer or endometrial cancer. In some embodiments, a score of at least 3, at least 3
homo (>75% uniformity), or at least 3 hetero % uniformity) indicates that the patient
will benefit from administration of an active agent comprising an OLRl antibody or
antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer,
endometrial cancer, or ovarian cancer.
In some ments, an H-score of at least 50 indicates that the patient will
benefit from administration of an active agent comprising an anti-FOLRl antibody or
antigen-binding nt thereof. In some embodiments, an H-score of at least 75 indicates
that a t With ovarian cancer will benefit from administration of an active agent
comprising an anti-FOLRl antibody or antigen—binding fragment thereof. In some
embodiments, an H-score of at least 50 indicates that a patient With NSCLC will benefit from
administration of an active agent comprising an anti—FOLRl dy or antigen-binding
fragment thereof. In some embodiments, an H-score of at least 50 indicates that a patient
with endometrial cancer will benefit from administration of an active agent comprising an
anti-FOLRl antibody or antigen-binding nt thereof. In one embodiment, an H—score is
ined using the FOLRl-2.l antibody.
In some ments, at least 25% of FOLRl membrane expression in a
ovarian tumor sample with an intensity of at least 3 tes that the patient will benefit from
administration of an active agent comprising an anti-FOLR] antibody or antigen-binding
fragment thereof. In some embodiments, at least 25% of FOLRl membrane expression in an
NSCLC sample with an intensity of at least 2 indicates that the patient will benefit from
administration of an active agent sing an anti-FOLRl antibody or antigen-binding
nt f. In some embodiments, at least 25% of FOLRl membrane expression in an
endometrial tumor sample with an intensity of at least 2 indicates that the patient will benefit
from administration of an active agent comprising an anti-FOLRl antibody or antigenbinding
fragment thereof. In one embodiment, the expression score is deteremined using the
FOLRl-2.l antibody.
In some embodiments, a score of at least 1 indicates an increased expression
of FOLRl. In some embodiments, a score of at least 2, at least 2 homo (>75% mity),
or at least 2 hetero (25-75% uniformity) indicates a decreased dose of the active agent should
be administered. In some embodiments, the cancer is lung cancer or endometrial cancer. In
some embodiments, a score of at least 3, at least 3 homo (>75% mity), or at least 3
hetero (25-75% uniformity) indicates a decreased dose of the active agent should be
stered. In some embodiments, the cancer is lung cancer, endometrial cancer, or
ovarian cancer.
In some embodiments, a score of at least 1 indicates an increased expression
of FOLRl. In some embodiments, a score of at least 2, at least 2 homo (>75% uniformity),
or at least 2 hetero (25-75% uniformity) identifies the cancer as likely to respond to a low
dose anti-FOLRl treatment. In some embodiments, the cancer is lung cancer or endometrial
cancer. In some embodiments, a score of at least 3, at least 3 homo (>75% uniformity), or at
least 3 hetero (25-75% uniformity) identifies the cancer as likely to respond to a low dose
anti-FOLRl ent. In some ments, the cancer is lung cancer, endometrial cancer,
or n cancer.
In some embodiments, a score of at least 2, at least 2 homo (>75%
uniformity), or at least 2 hetero (25-75% uniformity) identifies the cancer as being sensitive
to treatment with an active agent comprising an anti—FOLRl antibody or antigen-binding
nt thereof. In some embodiments, the cancer is lung cancer or endometrial cancer. In
some embodiments, a score of at least 3, at least 3 homo (>75% mity), or at least 3
hetero (ZS-75% uniformity) identifies the cancer as being sensitive to treatment with an
active agent comprising an anti-FOLRl antibody or antigen-binding fragment thereof. In
some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian .
In some embodiments, an H—score of at least 50 identifies a cancer as being
sensitive to treatment with an active agent comprising an anti-FOLRl antibody or antigen-
binding fragment thereof. In some embodiments, an H-score of at least 75 identifies an
ovarian cancer as being ive to treatment with an active agent comprising an anti-FOLRl
antibody or antigen-binding fragment thereof. In some embodiments, an H-score of at least
50 identifies an NSCLC as being sensitive to treatment with an active agent comprising an
anti-FOLRl antibody or antigen—binding fragment thereof. In some embodiments, an H-
score of at least 50 identifies an trial cancer as being sensitive to treatment with an
active agent comprising an anti-FOLRl antibody or antigen-binding fragment thereof. In one
embodiment, an H-score is determined using the 2.l antibody.
In some embodiments, at least 25% of FOLRl membrane expression in a
ovarian tumor sample with an ity of at least 3 identifies the cancer as being sensitive to
treatment with an active agent comprising an anti-FOLRl antibody or antigen-binding
fragment thereof. In some embodiments, at least 25% of FOLRl membrane sion in an
NSCLC sample with an ity of at least 2 identifies the cancer as being sensitive to
treatment with an active agent comprising an anti-FOLRl antibody or antigen-binding
fragment f. In some embodiments, at least 25% of FOLRl membrane sion in an
endometrial tumor sample with an intensity of at least 2 fies the cancer as being
sensitive to ent with an active agent comprising an anti-FOLRl antibody or antigenbinding
nt thereof. In one embodiment, the expression score is deteremined using the
FOLRl-2.l antibody.
In some embodiments, the reference sample is a positive reference sample or a
negative reference sample. In some embodiments, the reference sample comprises cells, cell
pellets, or tissue.
In some embodiments, the antibody, antigen-binding nt thereof, or
polypeptide of comprises a detection reagent selected from the group consisting of: an
enzyme, a fluorophore, a radioactive label, and a luminophore. In some embodiments, the
detection reagent is selected from the group consisting of: biotin, digoxigenin, fluorescein,
tritium, and rhodamine.
In some embodiments, the cancer is a FOLRl positive cancer. In some
embodiments, the cancer is selected from the group consisting of ovarian, brain, breast,
uterine, endometrial, pancreatic, renal, and lung cancer. In some ments, the lung
cancer is non small cell lung cancer or bronchioloalveolar carcinoma. In some embodiments,
the ovarian cancer is lial ovarian cancer. In some embodiments, the ovarian cancer is
platinum resistant, relapsed, or refractory.
In some embodiments, FOLRl expression is detected using at least one
additional anti-FOLRI antibody or antigen-binding fragment thereof. In some embodiments,
FOLRl expression is measured using two anti-FOLR] antibodies or antigen-binding
fragments thereof. In some embodiments, at least one antibody or antigen-binding fragment
thereof is bound to a solid support. In some embodiments, at least one antibody or antigen-
binding fragment f is bound to a microtiter plate.
In some ments, at least one additional antibody or antigen-binding
fragment thereof comprises a detection agent. In some embodiments, the ion agent is a
chromogenic detection agent, a enic detection agent, an enzymatic detection agent, or
an ochemiluminescent detection agent. In some embodiments, the detection agent is
horseradish peroxidase (HRP).
In some embodiments, the ELISA is a sandwich ELISA.
In some embodiments, the active agent comprises the FOLRl antibody
huMovl9. In some ments, the active agent is an antibody maytansinoid conjugate
comprising the FOLRl antibody 9 (comprising a heavy chain variable region of SEQ
ID NO:45 and a light chain variable region of SEQ ID NO:47), the sinoid DM4, and
the cleavable sulfo-SPDB linker (IMGN853).
In some ments, a method for identifying a cancer as likely to respond
to treatment with an antibody maytansinoid conjugate comprising the FOLRl antibody
9, the maytansinoid DM4 and a sulfo-SPDB linker (IMGN853), comprises
measuring FOLRl using an antibody comprising a heavy chain comprising the amino acids
of SEQ ID N027 and a light chain comprising the amino acids of SEQ ID NO:28 in an IHC
assay, wherein a score of at least 2 hetero indicates the cancer is likely to responds to the
treatment.
In some embodiments, a method for identifying a cancer as likely to respond
to treatment with an antibody maytansinoid conjugate comprising the FOLRl antibody
huMovl9, the sinoid DM4 and a sulfo-SPDB linker 53), comprises
measuring FOLRl using an antibody comprising a heavy chain comprising the amino acids
of SEQ ID N027 and a light chain comprising the amino acids of SEQ ID N028 in an IHC
assay, wherein a score of at least 1 indicates the cancer is likely to responds to the treatment.
In some embodiments, an article of manufacture provided herein comprises a
therapeutic active agent comprising an anti—FOLRl antibody or antigen-binding fragment
thereof described herein, a container, and a package insert or label indicating that the active
agent can be used to treat a cancer terized by the sed sion of FOLRl. In
some embodiments, an article of manufacture provided herein comprises a therapeutic active
agent comprising an anti-FOLRI antibody or antigen-binding nt thereof described
herein, a container, and a package insert or label indicating that the active agent can be used
to treat a cancer characterized by the sion of FOLRl at a level of 2, or 3 measured
using an antibody, antigen—binding fragment thereof, polypeptide, or composition provided
herein. In some embodiments, the anti—FOLRl antibody of the active agent is conjugated to a
cytotoxin. In some embodiments, the package insert or label tes that the active agent
can be used to treat a cancer characterized by the expression of FOLRl at a level of at least 1.
In some embodiments, the e insert or label indicates that the active agent can be used
to treat a cancer characterized by the expression of FOLRl at a level of at least 2, at least 2
homo (>75% uniformity), or at least 2 hetero (25—75% uniformity). In some embodiments,
the cancer is lung cancer or endometrial cancer. In some ments, the package insert or
label indicates that the active agent can be used to treat a cancer characterized by the
expression of FOLRl at a level of at least 3, at least 3 homo (>75% uniformity), or at least 3
hetero (25-75% uniformity). In some ments, the cancer is lung cancer, endometrial
cancer, or ovarian cancer.
In some embodiments, a combination diagnostic and pharmaceutical kit
provided herein comprises an dy, antigen-binding fragment thereof, polypeptide, or
composition ed herein for use in diagnosis and an active agent comprising an anti-
FOLRl antibody or antigen-binding fragment thereof for use in therapy. In some
embodiments, the detection antibody is able to detect FOLRl expression by IHC. In some
embodiments, the detection antibody is able to detect FOLR] expression by ELISA. In some
embodiments, the anti-FOLRl antibody in the active agent is ated to a cytotoxin.
In some embodiments, a diagnostic kit provided herein comprises an antibody,
antigen-binding fragment f or polypeptide provided herein, a reagent for
histochemistry (IHC), and one or more standardized reference samples, wherein the
standardized reference samples comprise cells, cell pellets, or formalin fixed paraffin
embedded tissue samples, and wherein the one or more standardized referenced samples are
from non-FOLRl expressing, low-FOLRl expressing, or high FOLRl expressing cells, cell
pellets, or tissues.
In some embodiments, an immunoassay kit for detecting shed FOLRl in a
sample comprises: (a) an antibody, antigen—binding fragment thereof, polypeptide, or
composition provided herein, and (b) a detection reagent. In some embodiments, the kit
r comprises a solid support for the capture reagent. In some embodiments, the capture
reagent is immobilized on the solid support. In some embodiments, the capture reagent is
coated on a microtiter plate. In some embodiments, the detection reagent is a second FOLRl
antibody. In some embodiments, the detection reagent is detected using a species specific
antibody. In some ments, the kit further comprises a detection means for the detection
reagent. In some embodiments, the detection means is colorimetric. In some embodiments,
the kit r comprises a FOLRl polypeptide as an antigen standard. In some
embodiments, the FOLRl polypeptide is FOLRl—Fc.
Active agents are also provided herein. In some embodiments, an active agent
comprises an anti-FOLRl antibody or antigen—binding fragment thereof for use in a method
for ng cancer, wherein said active agent is administered to a subject having cancer,
wherein increased expression of FOLRl has been detected in a cancerous sample obtained
from said subject using an antibody, antigen-binding fragment thereof, polypeptide or
composition provided herein.
In some embodiments, an active agent comprises an anti-FOLRl dy or
antigen-binding fragment thereof for use in a method for treating cancer, comprising: (a)
determining a FOLRl sion score from a ion of FOLRl expression in a cancerous
sample obtained from the patient, wherein the detection is med using an antibody,
antigen-binding fragment thereof, polypeptide, or composition ed herein; and (b)
administering an active agent comprising an anti-FOLRl antibody or n-binding
fragment thereof to the patient if the score indicates the patient will benefit from
administration of the active agent.
In some embodiments, an active agent comprises an anti-FOLR] antibody or
antigen-binding fragment thereof for use in a method for treating , comprising: (a)
ining a FOLRl expression score from a detection of FOLRl expression in a cancerous
sample obtained from the patient, wherein the ion is performed using an antibody,
antigen-binding fragment thereof, polypeptide, or ition ed herein; and (b)
instructing a healthcare provider to ster an active agent comprising an anti-FOLRl
antibody or antigen-binding nt thereof to the patient if the score indicates the t
will benefit from administration of the active agent.
In some embodiments, an active agent comprises an anti-FOLRl antibody or
antigen-binding fragment thereof for use in a method for ng cancer, comprising: (a)
submitting a cancerous sample ed from a patient having cancer for determining a
FOLRl expression score from a detection of FOLRl expression using the antibody, antigen-
binding nt thereof, polypeptide, or composition provided ; and (b) administering
an active agent sing an anti-FOLRl antibody or antigen-binding fragment thereof to
the t if the score indicates the patient will benefit from administration of the active
agent.
In some embodiments, an active agent comprises an anti-FOLRl antibody or
antigen-binding fragment thereof for use in a method for treating cancer, comprising: (a)
detecting FOLRl expression in a cancerous sample obtained from said patient,wherein the
detection is performed using the antibody, antigen—binding fragment thereof, polypeptide, or
composition provided herein; (b) determining a FOLRl expression score for said cancerous
sample; and (c) administering an active agent comprising an anti-FOLRl antibody or antigen-
bidning fragment f to the patient if the score indicates the patient will benefit from
administration of the active agent.
In some embodiments, an active agent comprises an anti-FOLRl antibody or
antigen-binding fragment thereof for use in a method for treating cancer, comprising: (a)
stering to a t a fixed dose of an active agent comprising an anti-FOLRl antibody
or antigen-binding fragment thereof; (b) detecting the FOLRl expression level in a cancerous
sample obtained from the patient ve to the FOLRl level in a reference sample,wherein
the detection is performed using an antibody, antigen—binding fragment thereof, polypeptide,
or composition provided herein; and (c) increasing the amount or frequency of subsequent
fixed doses if the patient’s FOLRI level is elevated.
In some embodiments, an active agent comprises an anti-FOLRl antibody or
antigen-binding fragment thereof for use in a method for treating cancer, comprising the step
of optimizing the therapeutic n of said active agent sing: (a) administering an
increased dose of an active agent comprising an anti-FOLR] antibody or antigen-binding
fragment thereof to a subject having cancer wherein an increased expression of FOLRl in a
cancerours sample from said subject has been detected using the antibody, antigen-binding
fragment thereof, polypeptide, or composition ed herein; or (b) administering a
decreased dose of the active agent to a subject having cancer wherein a decreased expression
of FOLRl in a cancerous sample from said subject has been detected.
In some embodiments, an active agent comprises an anti-FOLRl antibody or
antigen-binding fragment thereof for use in a method for treating cancer, sing the step
of zing the therapeutic regimen of said active agent comprising: (a) detecting the level
of FOLRl expression in a cancerous sample from said subject using an antibody, antigen-
binding fragment thereof, polypeptide, or composition provided herein; (b) determining a
FOLRl expression score for said cancerous sample; and (c) administering an increased dose
of an active agent comprising an anti-FOLRl dy or n-binding fragment f to
the subject if the score is low or administering a decreased dose of the active agent to the
subject if the score is high.
In some embodiments, an active agent comprises an anti-FOLRl antibody or
antigen-binding fragment thereof for use in a method for treating cancer, wherein FOLRlexpressing
cancer cells in a cancer patient are decreased, wherein: (a) the FOLRl level in a
cancerous sample obtained from a patient is detected by comparing it to the FOLRl level in a
reference sample using an antibody, antigen—binding nt thereof, polypeptide, or
ition provided herein; and (b) a fixed dose of the active agent is administered to the
patient if the patient’s FOLRl level is elevated; wherein the administration of the active agent
decreases the number of expressing cancer cells in the patient.
In some ments, an active agent comprises an anti-FOLRl antibody or
antigen-binding fragment thereof for use in a method for ng cancer, wherein FOLRlexpressing
cancer cells in a cancer patient are decreased, wherein: (a) a fixed dose of the
active agent is administered to a patient having a ; (b) the FOLRl level in a cancerous
sample obtained from the patient is detected relative to the FOLRl level in a reference
sample using an antibody, antigen-binding fragment thereof, polypeptide, or composition
provided herein; and (c) the amount or frequency of subsequent fixed doses is increased if the
patient’s FOLRl level is elevated compared to the reference sample; wherein the
administration of the active agent decreases the number of FOLRl-expressing cancer cells in
the patient.
Anti-FOLRl antibodies and antigen-binding fragments thereof for uses is
s of monitoring and methods of diagnosing are also provided herein. In some
embodiments, an anti-FOLRl antibody or n—binding fragment thereof for use in a
method for monitoring the therapeutic y of a fixed dose of the active agent in a patient
comprises: (a) detecting a first FOLRl level in a biological sample from a patient having
cancer using an antibody, antigen-binding fragment thereof, polypeptide, or composition
provided herein; (b) administering to the patient a fixed dose of the active agent; (c)
detecting a second FOLRl level in a biological sample from the patient following active
agent administration, wherein the detecting is med using an antibody, antigen-binding
fragment thereof, polypeptide, or ition provided herein; and (d) comparing the second
FOLRl level to the first FOLRl level; wherein a decrease n the first and second
FOLRl level indicates therapeutic efficacy.
In some embodiments, an anti-FOLRl antibody or antigen-binding fragment
thereof for use in a method for diagnosing whether a subject having cancer is likely to
d to a low dose anti-FOLRl treatment regimen, comprises: (a) contacting a biological
sample comprising cells from said cancer with an antibody, antigen-binding fragment f,
polypeptide, or composition provided herein; (b) detecting g of said antibody, antigen-
binding fragment, or polypeptide 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 cancer FOLRl level that is greater than the score for a
normal or low FOLRl expressing reference sample or a score for said cancer FOLRl level
that is equal to or r than the score for a high FOLRl expressing reference sample
identifies said cancer as likely to respond to a low dose of an active agent comprising an anti-
FOLRl antibody or antigen-binding fragment thereof.
In some embodiments, an anti-FOLRl antibody or antigen-binding fragment
thereof for use in a method for diagnosing r a cancer is sensitive to treatment with an
OLRl treatment, comprises: (a) detecting the level of FOLRl expression in a cancerous
sample from said cancer using an antibody, antigen—binding fragment thereof, polypeptide, or
ition provided herein, wherein said detecting comprises the use of a method that
distinguishes between staining intensity or staining uniformity in a FOLRl expressing
cancerous sample as compared to staining intensity or staining uniformity in one or more
nce samples; (b) determining a FOLRl staining intensity or staining uniformity score
for said cancerous sample; and (c) comparing the FOLRl staining intensity or staining
mity score determined in step (b) to a relative value determined by measuring FOLRl
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 ent with an active agent
comprising an anti-FOLRl antibody or n—binding fragment thereof and wherein a
FOLRl staining intensity score for said cancerous sample determined in step (b) that is
higher than said relative value identifies said cancer as being sensitive to treatment with the
active agent.
In some ments, an anti-FOLRl antibody or antigen-binding fragment
thereof for use in a method for diagnosing whether a cancer is sensitive to treatment with an
anti-FOLRl treatment, comprising: (a) ing the level of FOLRl sion in a
cancerous sample from said cancer using an antibody, antigen—binding fragment thereof,
ptide, or composition provided herein, wherein said detecting comprises the use of a
method that distinguishes between staining intensity or staining uniformity in a FOLRl
expressing cancerous sample as compared to staining intensity or ng mity in one
or more reference samples; (b) determining a FOLRl staining intensity or staining mity
score for said cancerous ; and (c) comparing the FOLRl staining intensity or staining
uniformity score determined in step (b) to a relative value determined by measuring FOLRl
n expression in at least one reference sample, wherein said at least one reference sample
is a tissue, cell, or cell pellet sample which is sensitive to treatment with an active agent
comprising an anti-FOLRl antibody or antigen—binding fragment thereof and wherein a
FOLRl staining intensity score for said cancerous sample determined in step (b) that is
higher than said relative value identifies said cancer as being sensitive to treatment with the
active agent.
In some embodiments, the use of the active agents or anti-FOLRl antibodies
or antigen-binding fragments thereof filI‘thCI' comprises administering an active agent
comprising an anti-FOLRl antibody or antigen—fragment thereof to the subject from whom
the cancerous sample or biological sample was obtained.
In some embodiments, the ous sample or biological sample is a bodily
fluid, cell, or tissue sample. In some embodiments, the cell is a circulating tumor cell. In
some embodiments, the bodily fluid is blood, ascites, urine, plasma, serum, or peripheral
blood.
In some embodiments of the active agents or anti-FOLRl antibodies or
antigen-binding fragmetns thereof, the detecting is by enzyme linked immunosorbent assay
(ELISA) and/or by immunohistochemistry (IHC). In some embodiments, the IHC is
calibrated IHC that can distinguish different levels of FOLRl expression. In some
embodiments, the IHC produces a range of staining intensity for samples having low cell
surface FOLRl expression, intermediate FOLRl cell surface sion, or high FOLRl cell
surface expression. In some ments, the IHC guishes between ng intensity
and staining uniformity in a FOLRl expressing cancerous sample or biological sample as
compared to a nce sample. In some embodiments, IHC is performed manually. In
some embodiments, the IHC is performed using an automated system. In some
embodiments, a FOLRl score is determined from the IHC. In some embodiments, the IHC
with an antibody or antigen-binding fragment described herein produces a range of staining
for cells that have increased FOLRI expression, particularly those within the level of staining
equal to or greater than 2.
In some embodiments, a score of at least 2 indicates that the patient will
benefit from administration of an active agent comprising an anti-FOLRl antibody or
antigen-binding fragment thereof. In some embodiments, a score of at least 2 homo (>75%
uniformity) or at least 2 hetero (25-75% uniformity) indicates that the patient will benefit
from administration of an active agent comprising an anti-FOLRl antibody or antigen-
binding fragment thereof. In some embodiments, the cancer is lung cancer or endometrial
cancer.
In some embodiments, a score of at least 3 indicates that the patient will
benefit from administration of an active agent sing an anti-FOLRl antibody or
antigen-binding nt thereof In some embodiments, a score of at least 3 homo (>75%
uniformity) or at least 3 hetero (25-75% uniformity) indicates that the patient will benefit
from administration of an active agent comprising an anti-FOLRl antibody or antigen-
g fragment thereof. In some embodiments, the cancer is lung cancer, endometrial
cancer, or ovarian cancer.
In some embodiments, a score of at least 2 indicates that the patient will
benefit from stration of an active agent comprising an anti-FOLRl antibody or
antigen-binding fragment thereof In some embodiments, a score of at least 2 homo (>75%
mity) or at least 2 hetero (25—75% uniformity) indicates that the patient will benefit
from administration of an active agent comprising an anti—FOLRl antibody or antigen-
g fragment thereof. In some ments, the cancer is lung cancer, trial
cancer, or ovarian cancer.
In some embodiments, a score of at least 2 indicates an a sed dose of the
active agent should be stered. In some embodiments, a score of at least 2 homo (>75%
mity) or at least 2 hetero (25-75% uniformity) indicates an a decreased dose of the
active agent should be administered. In some embodiments, the cancer is lung cancer,
endometrial canoe, or ovarian cancer.
In some embodiments, a score of at least 2 identifies the cancer as likely to
respond to a low dose anti-FOLRl treatment. In some embodiments, a score of at least 2
homo (>75% uniformity) or 2 hetero (25—75% mity) identifies the cancer as likely to
respond to a low dose anti-FOLRl treatment. In some embodiments, the cancer is lung
cancer or endometrial cancer.
In some embodiments, a score of at least 3 identifies the cancer as likely to
respond to a low dose anti-FOLRI treatment. In some embodiments, a score of at least 3
homo (>75% uniformity) or at least 3 hetero (25—75% uniformity) identifies the cancer as
likely to respond to a low dose OLRI treatment. In some embodiments, the cancer is
lung cancer, endometrial cance, or ovarian cancer.
In some embodiments, a score of at least 2 identifies the cancer as likely to
respond to a low dose anti-FOLRI treatment. In some embodiments, a score of at least 2
homo (>75% mity) or at least 2 hetero (25—75% uniformity) identifies the cancer as
likely to respond to a low dose anti—FOLRl treatment. In some embodiments, the cancer is
lung cancer, endometrial cance, or n cancer.
In some ments, a score of at least 2 identifies the cancer as being
ive to treatment with an active agent comprising an OLRl antibody or antigen-
binding fragment thereof. In some embodiments, a score of at least 2 homo (>75%
uniformity) or at least 2 hetero (25-75% uniformity) identifies the cancer as being sensitive
to treatment with an active agent comprising an anti-FOLRl antibody or antigen-binding
fragment thereof. In some embodiments, the cancer is lung cancer or endometrial cancer.
In some embodiments, a score of at least 3 identifies the cancer as being
sensitive to treatment with an active agent comprising an anti-FOLRl antibody or antigenbinding
fragment thereof. In some embodiments, a score of at least 3 homo (>75%
uniformity) or at least 3 hetero (25-75% mity) identifies the cancer as being sensitive
to treatment with an active agent comprising an anti-FOLRl dy or antigen-binding
fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cance, or
ovarian cancer.
In some embodiments, a score of at least 2 identifies the cancer as being
sensitive to treatment with an active agent comprising an anti-FOLR] dy or antigenbinding
fragment thereof. In some embodiments, a score of at least 2 homo (>75%
mity) or at least 2 hetero (25-75% uniformity) identifies the cancer as being sensitive
to treatment with an active agent comprising an anti—FOLRl antibody or antigen-binding
fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cance, or
ovarian cancer.
In some embodiments, a reference sample is a positive nce sample or a
negative reference sample. In some embodiments, the reference sample comprises cells, cell
s, or .
In some embodiments of the active agent or anti-FOLRl antibody or antigen-
binding fragment f for a use provided herein, the antibody, antigen-binding fragment,
or polypeptide provided herein further comprises a detection reagent selected from the group
consisting of: an enzyme, a fluorophore, a radioactive label, and a luminophore. In some
embodiments, the detection reagent is selected from the group consisting of: biotin,
genin, fluorescein, tritium, and rhodamine.
In some ments of the active agent or OLRl antibody or antigen-
binding fragment thereof for a use provided herein, the cancer is a FOLRl positive cancer. In
some embodiments, the cancer is selected from the group consisting of ovarian, brain, breast,
uterine, endometrial, pancreatic, renal, and lung cancer. In some ments, the lung
cancer is non small cell lung cancer or bronchioloalveolar oma. In some ments,
the ovarian cancer is epithelial ovarian cancer. In some embodiments, the n cancer is
platinum resistant, relapsed, or refractory.
In some embodiments of the active agent or anti-FOLRl antibody or antigen-
binding fragment thereof for a use provided herein, the FOLRl expression is detected using
at least one additional anti-FOLRl antibody or antigen-binding fragment thereof. In some
embodiments, the FOLRl expression is measured using two anti-FOLRl antibodies or
antigen-binding fragments thereof. In some embodiments, at least one antibody or antigen-
binding fragment thereof is bound to a solid support. In some embodiments, at least one
antibody or antigen-binding fragment thereof is bound to a microtiter plate. In some
embodiments, at least one antibody or antigen—binding fragment thereof ses a
detection agent. In some embodiments, the detection agent is a genic detection agent,
a fluorogenic detection agent, an enzymatic detection agent, or an electrochemiluminescent
ion agent. In some embodiments, the detection agent is horseradish peroxidase (HRP).
In some embodiments, the ELISA is a sandwich ELISA.
In some embodiments of the active agent or anti-FOLRl antibody or antigen-
binding fragment f for a use provided herein, the active agent comprises the FOLRl
dy huMovl9 or is the FOLRl antibody huMovl9. In some embodiments, the active
agent is administered as an antibody maytansinoid conjugate further comprising the
maytansinoid DM4 and the cleavable sulfo—SPDB linker (IMGN853).
In some embodiments, an dy, antigen-binding fragment, ptide, or
composition ed herein is for use as a diagnostic.
In some embodiments, an antibody, antigen-binding fragment, polypeptide, or
ition provided herein is for use in a method for diagnosing cancer in a patient
suffering therefrom. In some embodiments, the cancer is associated with elevated levels of
FOLRl.
In some embodiments, the binding affinity of an antibody, antigen-binding
fragment, or polypeptide is a binding affinity ed in Example 3 and/or shown in Figure
4, 5, and/or 6.
BRIEF DESCRIPTIONS OF THE DRAWINGS
Figure 1 provides images of IHC staining of NSCLC and ovarian
endometrioid adenocarcinoma samples using the 3532—1 and 3539-20 antibodies.
Figure 2 provides images of IHC staining of normal salivary gland and
pancreas samples using the 3532-1 and 3539—20 antibodies.
Figure 3 provides images of n blots of cell lysates using the 3539-21,
, 3533-8, and 3535-7 antibodies.
Figure 4 shows the binding of 3532-1, 3533-1, 3535-7, and 3539-21
antibodies to denatured KB cells (A) and non-denatured T47D cells (B) using a fluorescence
activated cell sorter (FACS).
Figure 5 shows the binding of 353.2-1, 353.3-1, 3535-7, and 3539-21
antibodies to recombinant human FOLR] using ELISA.
Figure 6 shows the binding of an anti-FOLR2 antibody and 353.2-1, 353.3-1,
7, and 3539-21 antibodies to FOLR2 (A) and the binding of an anti-FOLR3 antibody
and 3532-1, 3533-1, 3535-7, and 3539-21 antibodies to FOLR3 (B) by ELISA.
Figure 7 shows the binding of anti-FOLR] antibodies 2.1 and huMov19 to
deglycosylated and eated recombinant human FOLR] by ELISA.
Figure 8 shows the binding of anti-FOLR] dies 2.1, huMov19, and
BN3.2 to deglysolyated and non—treated s of KB and Igrov-l cells by western blot
analysis.
Figure 9 shows the relevant amino acids for resurfacing of the anti-FOLRl
FRIHCZ-l antibody and the kabat position corresponding to each residue.
Figure 10 shows the alignment of murine and humanized FRIHC2-1 antibody
ces for resurfacing. The murine heavy and light chain sequences correspond to SEQ
ID N027 and SEQ ID NO:28, respectively. The resurfaced humanized heavy chain
sequence corresponds to SEQ ID NO: 62, and the resurfaced human light chain version 1.0
and version 1.1 sequences correspond to SEQ ID NO:63 and SEQ ID NO:64, respectively.
The leader "S" in the light chain sequence (framework position -1) is not considered for
humanization and is not used in the zed antibody sequence, so it is not shown in the
figure.
Figure 11 shows the relevant amino acids for CDR grafting of the anti-FOLRl
FRIHC2-1 antibody and the kabat position corresponding to each residue.
Figure 12 shows the alignment of murine and humanized FRIHCZ-l
sequences for CDR grafting. The murine heavy and light chain sequences correspond to SEQ
ID N027 and SEQ ID NO:28, respectively. The grafted humanized heavy chain sequence
corresponds to SEQ ID NO: 65, and the grafted human light chain version 1.0 and version 1.1
sequences correspond to SEQ ID NO:66 and SEQ ID NO:67, respectively. The leader "S" in
the light chain sequence (framework position —1) is not considered for humanization and is
not used in the humanized antibody sequence, so it is not shown in the figure.
Figure 13 provides images of IHC staining of lung adenocarcinoma tissues
using the FOLR1-2.1 (353-21) antibody at varying dilutions.
Figure 14 provides images of IHC staining of ve normal tissue (fallopian
tube) (A) and cells (FOLRl transfected cells (B)) and negative cells nsfected cells (C))
using the FOLR1-2.1 (353-21) dy.
Figure 15 provides images of IHC staining of ovarian cancer tissue (A) and
lung adenocarcinoma tissue (B) samples using the FOLR1—2.1 (353-21) antibody.
Figure 16 shows membrane staining of tumor cells in an endometrial cancer
sample with the FOLR1-2.1 assay. The stromal cells are not stained.
Figure 17 shows a comparison of staining and scoring difference between (A)
the 2.1 assay and (B) the BN3.2 assay.
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure provides methods of ing human folate receptor 1
(FOLRl), including membrane FOLRl, shed FOLRl, and FOLRl on ating tumor cells,
and ing the efficacy of or the likelihood of response to the treatment of cancers
characterized by the overexpression of FOLRl. The detection methods can detect a ally
relevant dynamic range of FOLRl and therefore can be used for patient stratification, to
r or determine therapeutic efficacy, or the likelihood of response to the ent of
cancers characterized by the over expression of FOLRl. Novel FOLRl-binding
polypeptides, such as dies, that are useful in the FOLRl detection methods (e.g., IHC
for membrane bound and cell associated FOLRl) are also disclosed. Related ptides
and polynucleotides, itions comprising the FOLRl-binding agents, and methods of
making the FOLRl-binding agents are also provided.
1. Definitions
To facilitate an understanding of the present invention, a number of terms and
phrases are defined below.
The terms "human folate receptor 1," "FOLRl," or "folate receptor alpha (FR-
a)", as used herein, refers to any native human FOLRl, unless otherwise indicated. Thus, all
of these terms can refer to either a protein or nucleic acid sequence as indicated herein. The
term "FOLRl" encompasses "full-length," unprocessed FOLRl as well as any form of
FOLRl that s from processing within the cell. The term also encompasses naturally
occurring variants of FOLRl protein or nucleic acid, e.g., splice variants, allelic variants and
isoforms. The FOLRl polypeptides and polynucleotides 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 FOLRl sequences e, but
are not limited to NCBI reference numbers P15328, NP_001092242.l, AAX29268.l,
AAX37l 19.1, 937.l, and NP_057936.1.
The terms "shed antigen" and "shed FOLRl" are used interchangeably herein.
These terms refer to a FOLRl protein that is soluble and that is not cell associated. In some
embodiments it includes the extracellular domain (ECD) and the glycosylphosphatidyl
inositol (GPI) linker. In one embodiment, the shed FOLR] includes only the ECD. FOLRl
protein includes a signal peptide (amino acids 1-24), the FOLR] protein chain (amino acids
-233 or 234), and a propeptide which can be cleaved (amino acids 235 to 257). Mature
FOLRl protein lacks the signal peptide. Shed FOLRI can e amino acids 1 to 257, l to
233, l to 234, 25 to 233, 25 to 234. or any other fragments thereof. In some ments the
signal sequence is cleaved. In other embodiments the ECD and the GPI n can be
embedded in a membrane (e.g., a soluble lipid raft). In one embodiment, the shed FOLRl
can include amino acids l-233 or a fragment thereof.
The term ody" 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 h 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, chimeric antibodies,
humanized antibodies, human antibodies, fusion proteins comprising an n
determination portion of an antibody, and any other modified immunoglobulin molecule
comprising an n recognition site so long as the antibodies t the desired biological
activity. An antibody can be of any of the five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgGZ, IgG3, IgG4, IgAl and
IgA2), based on the identity of their heavy—chain constant domains referred to as alpha, delta,
epsilon, gamma, and mu, respectively. The different s of immunoglobulins have
different and well known subunit ures and three-dimensional configurations.
Antibodies can be naked or conjugated to other molecules such as , radioisotopes, etc.
In some embodiments, an antibody is a non-naturally occurring antibody. In
some embodiments, an antibody is purified from natural ents. In some embodiments,
an antibody is recombinantly produced. In some embodiments, an antibody is produced by a
hybridoma.
A "blocking" dy or an "antagonist" antibody is one which inhibits or
s biological activity of the antigen it binds, such as FOLRl. In a certain embodiment,
blocking antibodies or antagonist antibodies substantially or tely 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%.
The term "anti-FOLRI antibody" or "an antibody that binds to FOLRl" refers
to an antibody that is capable of binding FOLRl with sufficient affinity such that the
antibody is useful as a diagnostic and/or eutic agent in targeting FOLRl. Unless
otherwise specified, the extent of binding of an anti-FOLR] antibody to an unrelated, non-
FOLRl protein is less than about 10% of the binding of the dy to FOLRl as measured,
e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to
FOLRl has a dissociation constant (Kd) of 31 HM, 5100 nM, :10 nM, 51 nM, or 50.1 nM.
In one embodiment, the anti-FOLRI antibody does not bind FOLRZ, FOLR3, FOLR4, or
folic acid. Examples of FOLRl antibodies are known in the art and are disclosed in US.
Published Application Nos. 2012/0009181 and 2012/0282175 and US. Provisional
Application Nos. 61/695,791 and 61/756,254, and PCT publication W0201 1/106528, each of
which is herein incorporated by reference. The sequences of anti-FOLRl antibodies and
antigen-binding fragments thereof are provided in Tables 1-8.
The term "antibody fragment" refers to a n of an intact antibody and
refers to the antigenic ining variable regions of an intact dy. Examples of
antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments,
linear antibodies, single chain antibodies, and pecific antibodies formed from antibody
fragments. The term "antigen-binding fragment" of an antibody includes one or more
fragments of an antibody that retain the ability to specifically bind to an antigen. It has been
shown that the antigen—binding on of an antibody can be performed by certain
fragments of a full-length antibody. Examples of binding fragments assed within the
term "antigen-binding fragment" of an antibody e (without tion): (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains (e.g., an
antibody digested by papain yields three fragments: two antigen-binding Fab fragments, and
one PC nt that does not bind antigen); (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., an
dy digested by pepsin yields two fragments: a bivalent antigen-binding F(ab')2
fragment, and a ch' fragment that does not bind antigen) and its related F(ab') monovalent
unit; (iii) a Fd fragment consisting of the VH and CH1 domains (i.e., that portion of the heavy
chain which is ed in the Fab); (iv) a Fv fragment consisting of the VL and VH domains
of a single arm of an dy, and the d disulfide linked Fv; (v) a dAb (domain
antibody) or sdAb (single domain antibody) fragment (Ward et al., Nature 341:544-546,
1989), which consists of a VH domain; and (vi) an ed complementarity determining
region (CDR).
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 ent 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 oma, phage selection, recombinant
expression, and transgenic animals.
The term "humanized antibody" refers to forms of non-human (e.g., )
antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments
thereof that contain minimal man (e.g., murine) sequences. Typically, humanized
antibodies are human immunoglobulins in which residues from the mentary
determining region (CDR) are replaced by residues from the CDR of a non-human species
(e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability
(Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327;
Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the FV framework
region (FR) residues of a human globulin are replaced with the corresponding
residues in an antibody from a man species that has the desired specificity, affinity,
and lity. The humanized antibody can be further modified by the substitution of
onal residues either in the FV framework region and/or within the replaced non-human
residues to refine and optimize antibody specificity, affinity, and/or capability. In general,
the humanized dy will comprise ntially all of at least one, and typically two or
three, variable domains containing all or ntially all of the CDR regions that correspond
to the non-human immunoglobulin whereas all or substantially all of the FR regions are those
of a human immunoglobulin consensus sequence. The humanized antibody can also comprise
at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a
human immunoglobulin. Examples of methods used to generate humanized antibodies are
described in US. Pats. 5,225,539 and 641, Roguska et al., Proc. Natl. Acad. Sci., USA,
91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some
embodiments, a "humanized antibody" is a resurfaced dy. In some embodiments, a
"humanized antibody" is a CDR-grafted antibody.
A "variable region" of an antibody refers to the le region of the antibody
light chain or the variable region of the antibody heavy chain, either alone or in combination.
The le regions of the heavy and light chain each consist of four framework s
(FR) connected by three mentarity determining regions (CDRs) also known as
hypervariable regions. The CDRS in each chain are held together in close proximity by the
FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-
binding site of antibodies. There are at least two techniques for determining CDRs: (1) an
approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins
of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and
(2) an ch based on crystallographic studies of antigen-antibody complexes zikani
et al (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of these two approaches
are sometimes used in the art to determine CDRs.
The Kabat numbering system is generally used when referring to a residue in
the variable domain (approximately residues 1—107 of the light chain and residues 1-113 of
the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md. (1991)).
The amino acid position numbering as in Kabat, refers to the numbering
system used for heavy chain variable domains or light chain variable domains of the
compilation of antibodies in Kabat et al., Sequences of ns of Immunological Interest,
5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this
numbering system, the actual linear amino acid sequence can contain fewer or additional
amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable
domain. For example, a heavy chain le domain can include a single amino acid insert
ue 52a ing to Kabat) after residue 52 of H2 and inserted es (e.g., residues
82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat
numbering of residues can be determined for a given antibody by alignment at regions of
homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat
numbering convention varies between H32 and H34 depending on the length of the loop (this
is because the Kabat numbering scheme places the ions at H35A and H35B; if neither
35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if
both 35A and 35B are t, the loop ends at 34). The AbM hypervariable regions
represent a compromise between the Kabat CDRs and Chothia structural loops, and are used
by Oxford Molecular's AbM dy modeling software.
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The term "human antibody" means an antibody produced by a human or an
antibody having an amino acid sequence corresponding to an dy produced by a human
made using any technique known in the art. This definition of a human antibody includes
intact or ength antibodies, fragments thereof, and/or antibodies comprising at least one
human heavy and/or light chain polypeptide such as, for example, an antibody sing
murine light chain and human heavy chain polypeptides.
The term "chimeric antibodies" refers to antibodies wherein the amino acid
sequence of the immunoglobulin molecule is derived from two or more s. Typically,
the variable region of both light and heavy chains corresponds to the variable region of
antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the
desired specificity, affinity, and capability while the constant regions are homologous to the
sequences in antibodies derived from r (usually human) to avoid eliciting an immune
response in that s.
The terms "epitope" or "antigenic inant" 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, es can be formed
both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary
folding of a n. Epitopes formed from contiguous amino acids are typically retained
upon protein ring, 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-
amino acids in a unique spatial conformation.
"Binding affinity" lly 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 lly be represented by the dissociation constant (Kd) or the half-maximal effective
concentration (EC50). Affinity can be measured by common s 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 n 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 t invention. c illustrative
embodiments are described herein.
"Or " 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. In
one embodiment, the antibody's y as determined by a Kd will be between about 10'3 to
about 10'12 M, between about 10'6 to about 10'“ M, n about 10'6 to about 10'10 M,
between about 10'6 to about 10'9 M, between about 10'6 to about 10'8 M, or between about 10'
to about 10'7 M.
The phrase antially similar," or "substantially the same," as used ,
denotes a sufficiently high degree of similarity between two numeric values (generally one
associated with an dy of the ion 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.
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-FOLRl
antibody or nt thereof) and is defined by a generic a: A-L-C, wherein C =
cytotoxin, L = linker, and A = cell binding agent or anti-FOLRl antibody or antibody
fragment. Immunoconjugates can also be defined by the generic formula in reverse order: C-
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-FOLRl
antibody or a fragment thereof in a stable, covalent manner. Linkers can be susceptible to or
be substantially resistant to acid-induced ge, light-induced cleavage, peptidase-induced
cleavage, esterase-induced cleavage, and disulfide bond ge, at conditions under which
the compound or the antibody remains . Suitable linkers are well known in the art and
include, for example, disulfide groups, thioether groups, acid labile groups, photolabile
, peptidase labile groups and esterase labile groups. Linkers also include d
linkers, and hilic forms thereof as described herein and know in the art.
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, cells
or itions 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, cleotide,
vector, cell, or composition which is ed is substantially pure.
As used herein, "substantially pure" refers to material which is at least 50%
pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or
at least 99% pure.
The terms "elevated" FOLRl, "increased expression" of FOLRl and
"overexpression" of FOLRl refer to a sample which contains elevated levels of FOLRl
expression. The FOLRl can be elevated, increased, or overexpressed as compared to a
control value (e.g., expression level in a biological sample, tissue, or cell from a t
without , a sample or cancer known to express no or low FOLRl, a normal sample, or
a cancer that does not have elevated FOLR] values). For e, a sample with increased
expression can contain an increase of at least 2—fold, at least 3—fold, or at least 5-fold relative
to a control values.
FOLRl expression can be measured by immunohistochemistry and given a
ng intensity score or a staining uniformity score by ison to 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 ity of 2 is given to the test
sample if the intensity is comparable to the level 2 calibrated control). For example, a score
of l, 2, or 3 (3+), preferably a score of 2, or 3 (3+), by immunohistochemistry indicates an
increased expression of FOLRl. A staining uniformity that is heterogeneous or
homogeneous is also indicative of FOLRl 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.). See Table ll. In another example, an increase in FOLRl 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 FOLRl values).
A "reference " can be used to ate and compare the results
obtained in the s of the invention from a test sample. Reference samples can be cells
(e.g., cell lines, cell pellets) or tissue. The FOLRl levels in the "reference " can 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 FOLR]. A "reference sample" can also serve as a
baseline of FOLRl expression to which the test sample is compared. The "reference sample"
can include a prior sample or ne sample from the same patient, a normal reference with
a known level of FOLRl expression, or a reference from a nt patient population with a
known level of FOLRl expression. FOLRl levels can also be expressed as values in a
standard curve. A standard curve is a quantitative method of plotting assay data to determine
the concentration of FOLRl in a sample. In one ment, a reference sample is an
antigen standard comprising purified FOLRl or FOLRl-Fc. The diagnostic methods of the
invention can involve a comparison between expression levels of FOLRl in a test sample and
a "reference value." In some embodiments, the reference value is the expression level of the
FOLRl in a reference sample. A reference value can be a predetermined value and can also
be ined from reference samples (e.g., l biological samples or reference samples)
tested in el 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 al. 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 s or values are described herein and are also described in Examples
1 and Examples 8-10 of
In some embodiments, the reference sample is a sample from a healthy tissue,
in particular a corresponding tissue which is not affected by cancer or a corresponding tissue
which is not affected by a cancer that overexpresses FOLRl or a corresponding healthy tissue
that is known not to express detectable levels of FOLR. These types of reference samples are
ed to as negative control samples or "normal" reference samples. In other
embodiments, the nce sample is a sample from a tumor or y tissue that expresses
detectable FOLRl. These types of reference samples are referred to as positive control or
ve reference samples. Positive control samples can also be used as a comparative
indicator for the type (hetero versus homo) and/or degree (0, l, 2, 3) of staining intensity,
which correlates with the level of FOLRI expression. Positive control comparative samples
are also referred to as calibrated reference samples. Low or non-FOLRl expressing
references are bed herein in the es and also include all structures of the
esophagus, acinar cells/islets of the pancreas, interalveolar connective tissue of lung, and
acinar cells of the ry gland. For cell lines, exemplary non-expressors include BXPC3,
Panc-l, and ASPCI. Positive FOLRI references are described herein, for example, in the
Examples and also include ducts of pancreas, respiratory epithelium of normal lung, and
intercalated ducts of the salivary gland. In some embodiments, positive FOLRl references
include ducts of pancreas and intercalated ducts of the salivary gland. For cell lines,
exemplary high FOLRl expressors are described herein, for example, in the Examples and
also include KB, HeLa, 300.19 cells transfected with FOLRl, Igrov-l, and Wish, and
ary low FOLRl expressors include 3, Caov-3, SW620, T47D, and Skov-3.
Another positive high FOLRl reference is a cell line stably or transiently transfected with
FOLRl. Additional positive and negative samples for FOLRl are described in Table 13.
Appropriate positive and negative reference levels of FOLRl for a particular cancer can be
determined by measuring levels of FOLRl in one or more appropriate subjects, and such
reference levels can be ed to specific populations of subjects (e.g., a nce level can
be age-matched so that comparisons can be made between FOLRl levels in samples from
subjects of a certain age and reference levels for a particular disease state, phenotype, or lack
f in a certain age group). Such reference levels can also be tailored to specific
techniques that are used to measure levels of FOLRl in biological samples (e.g.,
assays, etc.).
As used herein, "immunohistochemistry" refers to histochemical and
immunologic methods used to e, for example, cells or tissues. Thus, the terms
"immunohistochemistry," "immunocytochemistry," and "immunochemistry" are used
interchangeably.
The term "primary antibody" herein refers to an antibody that binds
specifically to the target protein antigen in a sample. A primary antibody is generally the first
antibody used in an ELISA assay or IHC ure. In one embodiment, the primary
antibody is the only antibody used in an IHC ure. The term "secondary antibody"
herein refers to an antibody that binds specifically to a primary dy, thereby forming a
bridge or link between the primary dy and a subsequent reagent, if any. The secondary
antibody is generally the second antibody used in an immunohistochemical procedure.
A "sample" or "biological sample" of the present invention is of biological
origin, in specific embodiments, such as from eukaryotic organisms. In some embodiments,
the sample is a human , but animal samples may also be used. Non-limiting sources of
a sample for use in the t invention include solid tissue, biopsy tes, ascites, fluidic
extracts, blood, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin,
respiratory, inal, and genitourinary tracts, tears, saliva, milk, tumors, organs, cell
cultures and/or cell culture constituents, for example. A "cancerous sample" is a sample that
contains a cancerous cell. The method can be used to examine an aspect of expression of
FOLRl or a state of a , 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.
For the purposes herein, a "section" of a tissue sample s 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 n or section of tissue
comprises a homogeneous population of cells. In other cases, the selected portion comprises a
region of , e.g. the lumen as a non—limiting example. The selected portion can be as
small as one cell or two cells, or could ent many thousands of cells, for example. In
most cases, the collection of cells is ant, 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).
As used herein, the term re reagent" refers to a reagent capable of
binding and capturing a target molecule in a sample such that under suitable condition, the
capture reagent-target molecule complex can be separated from the rest of the sample. In one
embodiment, the capture reagent is immobilized. In one embodiment, the capture reagent in a
ch assay is an antibody or a mixture of different antibodies against a target
As used herein, the term "detectable antibody" refers to an antibody that is
capable of being detected either ly through a label amplified by a detection means, or
indirectly through, e. g., another antibody that is labeled. For direct labeling, the antibody is
typically conjugated to a moiety that is detectable by some means. In one embodiment, the
detectable antibody is a biotinylated antibody.
As used herein, the term "detection means" refers to a moiety or technique
used to detect the presence of the able antibody and includes detection agents that
y the immobilized label such as label ed onto a microtiter plate. In one
embodiment, the detection means is a fluorimetric detection agent such as avidin or
streptavidin.
Commonly a "sandwich ELISA" employs the ing steps: (1) microtiter
plate is coated with a capture dy; (2) sample is added, and any antigen present binds to
capture antibody; (3) detecting antibody is added and binds to antigen; (4) enzyme-linked
secondary antibody is added and binds to detecting antibody; and (5) ate is added and is
converted by enzyme to detectable form.
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.
By "correlate" or "correlating" is meant comparing, in any way, the
performance and/or results of a first analysis with the performance and/or s 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 s 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 ment, increased expression of FOLRl correlates
with increased hood of iveness of a FOLRl-targeting anti-cancer therapy.
The terms r" and "cancerous" refer to or describe the physiological
condition in mammals in which a population of cells are characterized by unregulated cell
growth. es of cancer include, but are not limited to, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia. More particular examples of such cancers include cancers of
elial, mesenchymal, or epithelial origin, such as lung cancer (e.g., squamous cell
cancer, small-cell lung cancer, non—small cell lung cancer, adenocarcinoma of the lung,
mesothelioma, and squamous carcinoma of the lung), cancer of the peritoneum (e. g., primary
peritoneal), hepatocellular cancer, gastrointestinal cancer, atic cancer, glioblastoma,
cervical cancer, ovarian cancer (serous or endometrioid), liver cancer, r cancer,
hepatoma, breast cancer, colon cancer, colorectal cancer, endometrioid (e.g., endometrial
adenocarcinoma) or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, brain cancer (e. g.
glioblastoma, tumors of the choroid plexus) and s types of head and neck cancers, and
also tumors of blood vessels and fallopian tubes. Cancers also ass cancers which
contain cells having elevated FOLRl expression levels. Such FOLRl-elevated cancers
include, but are not d to, ovarian, non-small cell lung cancer (adenocarcinoma), e,
trial, atic, renal, lung, and breast cancer.
"Tumor" and "neoplasm" refer to any mass of tissue that result from excessive
cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including
ncerous s.
The terms "cancer cell," "tumor cell," and grammatical equivalents refer to
the total population of cells derived from a tumor or a ncerous 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 ing solely to those tumor cells lacking the capacity to
renew and entiate to distinguish those tumor cells from cancer stem cells.
The term "subject" refers to any animal (e.g., a mammal), including, but not
limited to humans, non-human primates, rodents, and the like, which is to be the ent of
a ular treatment. Typically, the terms "subject" and "patient" are used interchangeably
herein in reference to a human subject.
The term "pharmaceutical formulation" refers to a preparation which is in such
form as to permit the biological ty 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.
An "effective amount" of an antibody or immunoconjugate 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.
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 ment, stop) tumor metastasis; inhibit, to some extent, tumor
growth; relieve to some extent one or more of the symptoms associated with the cancer;
and/or result in a favorable response such as sed progression-free survival (PFS),
disease-free survival (DFS), or overall survival (OS), complete response (CR), partial
response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD),
a d time to progression (TTP), a decrease in CA125 in the case of ovarian cancer, or
any combination thereof. See the definition herein of ing." To the extent the drug can
prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In
certain embodiments, fication of increased FOLRl levels allows for stration of
decreased amounts of the FOLRl-targeting therapeutic to achieve the same therapeutic effect
as seen with higher dosages. A "prophylactically effective amount" refers to an amount
ive, at dosages and for s of time necessary, to achieve the desired prophylactic
result. Typically but not arily, since a prophylactic dose is used in subjects prior to or
at an r stage of disease, the prophylactically effective amount will be less than the
eutically effective amount.
The term "respond favorably" generally refers to causing a beneficial state in a
subject. With respect to cancer treatment, the term refers to providing a therapeutic effect on
the subject. Positive therapeutic effects in cancer can be measured in a number of ways (See,
W.A. Weber, J. Nucl. Med. 50zlS-IOS (2009)). For example, tumor growth inhibition,
lar 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 NC] standards, a T/C S 42% is the minimum level of
anti-tumor activity. A T/C <lO% is considered a high anti-tumor activity level, with T/C (%)
= Median tumor volume of the treated / Median tumor volume of the control x 100. A
favorable response can be assessed, for example, by increased ssion-free survival
(PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial
response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD),
a reduced time to progression (TTP), a decrease in CA125 in the case of ovarian cancer or
any ation thereof.
PPS, DFS, and OS can be measured by standards set by the National Cancer
Institute and the US. Food and Drug Administration for the approval of new drugs. See
Johnson et al, (2003) J. Clin. Oncol. 21(7):]404-1411.
"Progression free survival" (PFS) refers to the time from enrollment to disease
progression or death. PFS is generally measured using the Kaplan-Meier method and
Response Evaluation ia in Solid Tumors T) l.l standards. Generally,
progression free survival refers to the situation wherein a patient remains alive, without the
cancer getting worse.
"Time to Tumor ssion" (TTP) is defined as the time from enrollment to
disease progression. TTP is generally measured using the RECIST l.l criteria.
A "complete response" or "complete remission" or "CR" indicates the
disappearance of all signs of tumor or cancer in response to treatment. This does not always
mean the cancer has been cured.
A al response" or "PR" refers to a decrease in the size or volume of one
or more tumors or lesions, or in the extent of cancer in the body, in response to treatment.
"Stable disease" refers to disease without progression or relapse. In stable
disease there is neither sufficient tumor shrinkage to qualify for partial response nor sufficient
tumor increase to qualify as progressive disease.
"Progressive disease" refers to the appearance of one more new s or
tumors and/or the unequivocal progression of existing non-target lesions. Progressive disease
can also refer to a tumor growth of more than 20 percent since treatment began, either due to
an increases in mass or in spread of the tumor.
"Disease free survival" (DFS) refers to the length of time during and after
treatment that the t remains free of disease.
ll Survival" (OS) refers to the time from patient enrollment to death or
censored at the date last known alive. OS includes a prolongation in life expectancy as
compared to naive or untreated individuals or patients. l survival refers to the situation
wherein a patient remains alive for a defined period of time, such as one year, five years, etc.,
e.g., from the time of sis or treatment.
A "decrease in CAl25 levels" can be assessed according to the Gynecologic
Cancer roup (GCIG) guidelines. For e, CAl25 levels can be measured prior to
treatment to establish a baseline CA125 level. CA125 levels can be measured one or more
times during or after treatment, and a reduction in the CA125 levels over time as compared to
the ne level is ered a decrease in CA125 levels.
Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to
alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt
ssion of a diagnosed pathologic condition or disorder. Thus, those in need of treatment
include those already diagnosed with or suspected of having the disorder. In certain
embodiments, a subject is successfully "treated" for cancer according to the s of the
present invention if the patient shows one or more of the following: a reduction in the number
of or te absence of cancer cells; a reduction in the tumor size; inhibition of or an
absence of cancer cell ation into peripheral organs including, for example, the spread of
cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or
an absence of tumor growth; relief of one or more symptoms associated with the specific
cancer; d morbidity and ity; improvement in quality of life; ion in
tumorigenicity, tumorigenic frequency, or genic capacity, of a tumor; reduction in the
number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a
non-tumorigenic state; increased progression-free survival (PFS), disease-free survival
(DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease
(SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), a decrease
in CA125 in the case of ovarian cancer, or any combination thereof.
lactic or preventative measures refer to therapeutic measures that
prevent and/or slow the development of a ed pathologic condition or disorder. Thus,
those in need of prophylactic or preventative measures include those prone to have the
disorder and those in whom the disorder is to be prevented.
As used herein, the term "healthcare provider" refers to individuals or
institutions which directly ct with and administer to living subjects, e.g., human
ts. miting examples of healthcare providers include doctors, nurses, technicians,
therapist, pharmacists, counselors, alternative medicine practitioners, medical facilities,
doctor’s off1ces, hospitals, emergency rooms, clinics, urgent care centers, alternative
medicine clinics/facilities, and any other entity providing general and/or specialized
treatment, assessment, maintenance, therapy, medication, and/or advice ng to all, or any
portion of, a patient's state of health, ing but not limited to general medical, specialized
medical, surgical, and/or any other type of treatment, assessment, maintenance, therapy,
medication and/or advice.
In some aspects, a healthcare provider can ster or instruct r
healthcare provider to administer a therapy to treat a cancer. "Administration" of a therapy,
as used herein, includes prescribing a therapy to a subject as well as delivering, applying, or
giving the therapy to a subject. A healthcare provider can implement or instruct another
healthcare provider or patient to perform the following s: obtain a sample, process a
sample, submit a sample, receive a sample, transfer a sample, analyze or measure a sample,
quantify a , provide the results obtained after analyzing/measuring/quantifying a
sample, receive the results obtained after ing/measuring/quantifying a sample,
compare/score the results obtained after analyzing/measuring/quantifying one or more
samples, provide the comparison/score from one or more samples, obtain the
comparison/score from one or more samples, administer a y or therapeutic agent (e.g,
a FOLRl binding agent), commence the administration of a therapy, cease the administration
of a therapy, continue the administration of a therapy, temporarily upt the administration
of a therapy, increase the amount of an administered therapeutic agent, decrease the amount
of an stered therapeutic agent, continue the administration of an amount of a
therapeutic agent, increase the ncy of administration of a therapeutic agent, decrease
the frequency of administration of a therapeutic agent, maintain the same dosing ncy
on a therapeutic agent, replace a therapy or therapeutic agent by at least another therapy or
therapeutic agent, combine a therapy or therapeutic agent with at least another therapy or
additional therapeutic agent. These actions can be performed by a care provider
automatically using a computer—implemented method (e.g, via a web service or stand-alone
computer system).
"Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to
polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their s, or
any substrate that can be orated into a polymer by DNA or RNA rase. A
polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their
analogs. If present, modification to the nucleotide ure can be imparted before or after
assembly of the polymer. The ce of nucleotides can be interrupted by non-nucleotide
components. A polynucleotide can be further modified after polymerization, such as by
conjugation with a labeling component. Other types of modifications include, for example,
"caps," substitution of one or more of the naturally occurring nucleotides with an analog,
intemucleotide modifications such as, for example, those with uncharged es (e.g.,
methyl onates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged
linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant
moieties, such as, for example, proteins (e.g., ses, toxins, antibodies, signal peptides,
ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing
ors (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing
alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as
unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups,
ted by standard protecting groups, or ted to prepare onal linkages to
additional nucleotides, or can be conjugated to solid supports. The 5' and 3' terminal OH can
be phosphorylated or substituted with amines or organic capping group moieties of from 1 to
carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are
generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-
ribose, carbocyclic sugar analogs, alpha-anomeric , epimeric sugars such as
arabinose, xyloses or lyxoses, se sugars, fiaranose sugars, sedoheptuloses, acyclic
analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester
linkages can be replaced by alternative linking groups. These alternative linking groups
include, but are not limited to, ments wherein phosphate is replaced by P(O)S
("thioate"), P(S)S ("dithioate"), (O)NR2 ate"), P(O)R, P(O)OR', CO or CH2
("formacetal"), in which each R or R‘ is ndently H or tuted or unsubstituted alkyl
(1-20 C) optionally containing an ether (——O——) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl
or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description
applies to all polynucleotides referred to herein, including RNA and DNA.
The term "vector" means a construct, which is capable of ring, and
expressing, one or more ) or sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA expression vectors,
plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic
condensing agents, DNA or RNA expression vectors encapsulated in mes, and certain
eukaryotic cells, such as producer cells.
The terms "polypeptide, peptide," and "protein" are used interchangeably
herein to refer to rs of amino acids of any . The polymer can be linear or
branched, it can comprise modified amino acids, and it can be interrupted by non-amino
acids. The terms also encompass an amino acid polymer that has been modified naturally or
by intervention; for example, disulfide bond ion, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as conjugation with a
labeling component. Also included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It is understood that, because the
polypeptides of this ion are based upon antibodies, in n embodiments, the
polypeptides can occur as single chains or associated chains. In some ments, a
polypeptide, peptide, or protein is non—naturally occurring. In some ments, a
polypeptide, peptide, or protein is purified from other naturally occurring components. In
some embodiments, the polypeptide, peptide, or protein is recombinantly produced.
The terms "identical" or percent "identity" in the t of two or more
nucleic acids or ptides, refer to two or more sequences or subsequences that are the
same or have a ed percentage of nucleotides or amino acid residues that are the same,
when compared and aligned (introducing gaps, if necessary) for maximum correspondence,
not considering any conservative amino acid substitutions as part of the sequence identity.
The percent identity can be measured using sequence comparison software or algorithms or
by visual inspection. s algorithms and software are known in the art that can be used
to obtain alignments of amino acid or nucleotide sequences. One such non-limiting example
of a sequence alignment algorithm is the algorithm described in Karlin et a1, 1990, Proc.
Natl. Acad. Sci, 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci,
90:5873-5877, and incorporated into the NBLAST and XBLAST ms (Altschul et al.,
1991, Nucleic Acids Res., 25:3389—3402). In certain ments, Gapped BLAST can be
used as bed in Altschul et al., 1997, c Acids Res. 25:3389-3402. BLAST-2, WU-
BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2
(Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional
publicly ble software programs that can be used to align sequences. In certain
embodiments, the percent identity between two nucleotide sequences is determined using the
GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of
40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative
embodiments, the GAP m in the GCG software package, which incorporates the
thm of Needleman and Wunsch (J. M01. Biol. (48):444-453 (1970)) can be used to
determine the percent ty between two amino acid sequences (e.g., using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a
length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent ty
between nucleotide or amino acid sequences is determined using the algorithm of Myers and
Miller (CABIOS, 4: 1 1-17 (1989)). For example, the percent identity can be determined using
the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length
penalty of 12 and a gap penalty of 4. Appropriate parameters for maximal alignment by
particular alignment software can be determined by one skilled in the art. In certain
embodiments, the default parameters of the alignment software are used. In certain
ments, the percentage identity "X" of a first amino acid sequence to a second
sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid
residues scored as identical matches in the alignment of the first and second sequences (as
aligned by visual inspection or a particular sequence alignment program) and Z is the total
number of residues in the second ce. If the length of a first sequence is longer than the
second sequence, the percent identity of the first sequence to the second sequence will be
longer than the percent identity of the second sequence to the first sequence.
As a non-limiting example, whether any particular polynucleotide has a
n tage sequence identity (e.g., is at least 80% identical, at least 85% identical, at
least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99%
identical) to a reference sequence can, in certain embodiments, be determined using the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 e Drive, n, WI 53711).
Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2: 482 489 (1981), to find the best segment of homology between two
sequences. When using Bestfit or any other sequence alignment program to determine
whether a particular sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set such that the percentage of identity
is calculated over the full length of the reference nucleotide sequence and that gaps in
homology of up to 5% of the total number of tides in the reference sequence are
allowed.
In some embodiments, two nucleic acids or polypeptides of the ion are
substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide
or amino acid residue identity, when compared and aligned for maximum correspondence, as
measured using a ce comparison algorithm or by visual inspection. In certain
embodiments, identity exists over a region of the sequences that is at least about 10, about 20,
about 40-60 residues in length or any integral value therebetween, or over a longer region
than 60-80 es, at least about 90-100 residues, or the sequences are substantially
identical over the full length of the ces being compared, such as the coding region of a
nucleotide ce for example.
A "conservative amino acid substitution" is one in which one amino acid
residue is replaced with another amino acid residue having a r side chain. Families of
amino acid residues having similar side chains have been defined in the art, including basic
side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., gine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, e,
phenylalanine, methionine, tryptophan), ranched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, phan, histidine). For
example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In
certain embodiments, conservative tutions in the sequences of the polypeptides and
antibodies of the invention do not abrogate the binding of the polypeptide or antibody
containing the amino acid sequence, to the antigen(s), i.e., the FOLRl to which the
polypeptide or antibody binds. Methods of fying nucleotide and amino acid
conservative substitutions which do not eliminate n-binding are well— known in the art
(see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et a1. Protein Eng.
12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).
As used in the present disclosure and claims, the singular forms "a," "an," and
"the" e plural forms unless the context clearly es otherwise.
It is understood that wherever embodiments are described herein with the
language "comprising," otherwise ous embodiments described in terms of "consisting
of" and/or "consisting essentially of" are also provided.
The term r" 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. FOLRl ng agents
The present invention provides agents that specifically bind human FOLRl.
These agents are referred to herein as "FOLRl-binding agents." In certain embodiments, the
FOLRl binding agents are antibodies, immunoconjugates or polypeptides. The amino acid
and nucleotide sequences for human FOLRl are known in the art and are also provided
herein as represented by SEQ ID NO:1 and SEQ ID NO:2.
Human Folate Receptor 1:
MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLH
EQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYEC
SPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWT
SGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQG
NPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS (SEQ ID NO: 1)
Human Folate Receptor 1 Nucleic Acid ce:
atggctcagcggatgacaacacagctgctgctccttctagtgtgggtggctgtagtaggggaggctcagacaaggattgcatgggcc
aggactgagcttctcaatgtctgcatgaacgccaagcaccacaaggaaaagccaggccccgaggacaagttgcatgagcagtgtcg
accctggaggaagaatgcctgctgttctaccaacaccagccaggaagcccataaggatgtttcctacctatatagattcaactggaacc
actgtggagagatggcacctgcctgcaaacggcatttcatccaggacacctgcctctacgagtgctcccccaacttggggccctggat
ccagcaggtggatcagagctggcgcaaagagcgggtactgaacgtgcccctgtgcaaagaggactgtgagcaatggtgggaagat
tgtcgcacctcctacacctgcaagagcaactggcacaagggctggaactggacttcagggtttaacaagtgcgcagtgggagctgcc
cctttccatttctacttccccacacccactgttctgtgcaatgaaatctggactcactcctacaaggtcagcaactacagccgag
ggagtggccgctgcatccagatgtggttcgacccagcccagggcaaccccaatgaggaggtggcgaggttctatgctgcagccatg
agtggggctgggccctgggcagcctggcctttcctgcttagcctggccctaatgctgctgtggctgctcagc (SEQ ID N02)
Thus, in some embodiments, the FOLRI binding agents can bind to an epitope
of SEQ ID NO:1.
In some embodiments, an anti—FOLRl antibody can specifically binds to an
epitope of FOLRl (SEQ ID NO: 1), wherein epitope comprises an N—glycosylated amino acid.
Such antibodies will therefore bind to FOLRl when it is glycosylated and will not bind to
FOLRl when it is not glycosylated. In other words, the binding of these antibodies is glycol-
dependent. These antibodies are advantageous in that they can be used to distinguish
n glycosylated and non-glycosylated forms of FOLRl. Given that glycosylation can
be required for membrane localization, the antibodies can advantageously be used for
membrane specific ng.
In some embodiments, the anti—FOLRl antibody can specifically bind to an
epitope of FOLRl comprising N—glycosylated amino acid 69 of FOLRl. In some
embodiments, the anti-FOLRl antibody can specifically bind to an epitope of FOLRl
comprising N—glycosylated amino acid 161 of FOLRl. In some embodiments, the anti-
FOLRl antibody can specifically bind to an epitope of FOLRl comprising N—glycosylated
amino acid 201 of FOLRl.
In certain embodiments, the anti-FOLRI antibody is the antibody produced by
the hybridoma deposited with the American Type e Collection , located at
10801 University Boulevard, Manassas, VA 20110 on April 16, 2013 under the terms of the
Budapest Treaty and having ATCC deposit no. PTA-120196 ("FOLRl-9.20," also referred to
as "IMGN 0," 20," or "9.20"). In certain embodiments, the anti-FOLRl
antibody is the antibody produced by the hybridoma deposited with the ATCC on April 16,
2013 and having ATCC t no. PTA—120197 ("FOLRl-2.l," also referred to as "IMGN
3532-1," "3532-1," "2.1," or "muFRIHCZ-l").
The FOLRl-binding agents include FOLRl-binding agents that comprise the
heavy and light chain CDR sequences of (i) C2-1, which is also known as -
2.1," "IMGN 3532-1," "3532-1," or "2.1", (ii) muFRIHC5-7, which is also known as
"IMGN 353.5-7," "353.5-7" or "5.7," (iii) " muFRIHC9-20," which is also known as
"FOLRl-9.20," "IMGN 3539-20," 20," or "9.20," (iv) resurfaced huFRIHC2-1
n 1.0 or 1.01, or (v) CDR grafted huFRIHC2-1 version 1.0 or 1.01, which are provided
in Tables 1 and 2 below. The FOLRl-binding agents also include FOLRl-binding agents
that comprise the heavy and light chain CDR sequences of the ite CDRs provided in
Tables 1 and 2 below.
Table 1: Variable heavy chain CDR amino acid sequences
muFRIHC2-1 NSYIH (SEQ WIYPESLNTQYNEKFKA RGIYYYSPYALDH
muFRIHC5-7 NYYIH WIYPGSFNVEYNEKFKA RGIYFYSPYALDY
muFRIHC9-20 NYYIH WIYPENVNVRYNDKFKA RGIYYYSPYAMDY
("9.20") (SEQ ID (SEQ ID NO:16) (SEQ ID NO:17)
NO:15)
Composite N(Y/S)YIH WIYP(G/E)(S/N)(F/V/L)N(V/ RGIY(F/Y)YSPYA(L/
(SEQ ID T)(E/R/Q)YN(E/D)KFKA M)D(Y/H) (SEQ ID
NO:21) (SEQ ID NO:22) NO:23)
Table 2: Variable light chain CDR amino acid sequences
muFRIHC2-1 KSSKSLLNSDGFTYLD LVSNHFS (SEQ ID FQSNYLPLT
muFRIHC5-7 KSTESLLNSDGFTYLD LVSNHFS (SEQ ID FQSNYLPLT
muFRIHC9-20 KSTKSLLNSDGFTYLD LVSNHFS (SEQ ID PLT
Composite KS(T/S)(K/E)SLLNSDGFTY LVSNHFS (SEQ ID FQSNYLPLT
The FOLRl binding les can be antibodies or antigen g fragments
that specifically bind to FOLRl that comprise the CDRs of antibody 2.1 (i.e., SEQ ID NOS:
3-8), 5.7 (i.e., SEQ ID NOS: 9-14), or 9.20 (i.e., SEQ ID NOS: 15-20), with up to four (i.e., 0,
l, 2, 3, or 4) conservative amino acid substitutions per CDR. The FOLRl binding molecules
can be antibodies or antigen-binding fragments that cally bind to FOLRl that comprise
the CDRS of the composite sequence shown above (i.e., SEQ ID NOS: 21-26), with up to four
(i.e., 0, l, 2, 3, or 4) conservative amino acid substitutions per CDR.
The FOLRl binding molecules can be antibodies or antigen-binding fragments
that specifically bind to FOLRI that comprise the CDRs of antibody produced by the
hybridoma ofATCC deposit no. PTA-120196 or 0197.
Polypeptides can comprise one of the individual variable light chains or
variable heavy chains described herein. Antibodies and polypeptides can also comprise both
a le light chain and a variable heavy chain. The variable light chain and le heavy
chain sequences of murine antibodies 2.1, 5.7, and 9.20 and humanized 2.1 are provided in
Tables 3 and 4 below.
Table 3: le heavy chain amino acid sequences
Antibody VH Amino Acid Sequence (SEQ ID NO)
l’nuFRIHCZ-l
QVQLQQSGPELVKPGASVRISCKASGYTFTNSYIHWVKKRPGQGL
("2.1")
EWIGWIYPESLNTQYNEKFKAKATLTADKSSSTSYMQLSSLTSEDS
AVYFCARRGIYYYSPYALDHWGQGASVTVSS (SEQ ID NO:27)
muFRIHC5-7 QVQLQQSGPEVVKPGASVRISCKASGYTFTNYYIHWVKQRPGQGL
EWIGWIYPGSFNVEYNEKFKAKATLTADKSSSTVYMQLSSLTSEDS
("5' 7,,) AVYFCARRGIYFYSPYALDYWGQGASVTVSS (SEQ ID NO:29)
muFRIHC9-20 SGPDLVKPGASVRISCKASGFTFTNYYIHWVKQRPGQGL
EWIGWIYPENVNVRYNDKFKAKATLTADKSSSTAYMQLSSLTSED
("9 20")‘
SAVYFCARRGIYYYSPYAMDYWGQGASVTVSS (SEQ ID NO:31)
huFRIHC2-l QVQLVQSGAEVVKPGASVKISCKASGYTFTNSYIHWVKKRPGQGL
EWIGWIYPESLNTQYNQKFQGKATLTADKSSSTSYMQLSSLTSEDS
(resurfaced)
AVYFCARRGIYYYSPYALDHWG O GASVTVSS SE 0 ID NOI62
huFRIHC2-l QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSYIHWVRQAPGQG
LEWMGWIYPESLNTQYNEKFKARVTMTRDTSISTAYMELSRLRSD
(grafted)
DTAVYYCARRGIYYYSPYALDHWGQGTLVTVSSAST (SEQ ID
Table 4: Variable light chain amino acid sequences
Antibody VL Amino Acid Sequence (SEQ ID NO)
muFRIHC2-l SDVVLTQTPLSLPVNIGDQASISCKSSKSLLNSDGFTYLDWYLQKPG
QSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
("2 l")'
FQSNYLPLTFGGGTKLEIKR (SEQ ID NO:28)
C5-7 SDVVLTQTPLSLPVNIGDQASISCKSTESLLNSDGFTYLDWYLQKPG
QSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
("5 7,)‘
FQSNYLPLTFGGGTKLEVKR (SEQ ID NO:30)
muFRIHC9-20 QTPLSLPVNLGDQASISCKSTKSLLNSDGFTYLDWYLQKP
GQSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYY
(,9 20,)'
CFQSNYLPLTFGGGTKLEIKR (SEQ ID NO:32)
huFRIHCZ-l V. DVVLTQSPLSLPVNLGQPASISCRSSRSLLNSDGFTYLDWYLQKPGQ
SPRLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCF
1 0 faced)
QSNYLPLTFGQGTKLEIKR SEQ ID NO:63
huFRIHC2-l v. DVVLTQSPLSLPVNLGQPASISCKSSKSLLNSDGFTYLDWYLQKPG
IYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
1 01 (resurfaced)'
F o SNYLPLTFG o GTKLEIKR SE 0 ID NO:64
huFRIHC2-l v. DIVMTQTPLSLSVTPGQPASISCRSSRSLLNSDGFTYLDWYLQKPGQ
SPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF
1 0 (grafted)'
QSNYLPLTFGQGTKLEIK SEQ ID NO:66
C2-l v. DIVMTQTPLSLSVTPGQPASISCKSSKSLLNSDGFTYLDWYLQKPGQ
SPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF
1. 01 ed)
QSNYLPLTFGQGTKLEIK SEQ ID NO:67
Also provided are polypeptides that comprise: (a) a polypeptide having at least
about 90% sequence identity to SEQ ID NOsz27, 29, 31, 62, or 65; and/or (b) a polypeptide
having at least about 90% sequence identity to SEQ ID NOsz28, 30, 32, 63, 64, 66, or 67. In
certain embodiments, the ptide comprises a polypeptide having at least about 95%, at
least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence
identity to SEQ ID NOs227-32 or 62-67. Thus, in certain embodiments, the polypeptide
comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID ,
29, 31, 62, or 65 and/or (b) a polypeptide having at least about 95% sequence identity to SEQ
ID NOS:28, 30, 32, 63, 64, 66, or 67. In certain embodiments, the polypeptide comprises (a)
a polypeptide having the amino acid sequence of SEQ ID NOsz27, 29, 31, 62, or 65; and/or
(b) a polypeptide having the amino acid sequence of SEQ ID NOsz28, 30, 32, 63, 64, 66, or
67. In certain embodiments, the polypeptide is an antibody and/or the polypeptide
specifically binds FOLRl. In certain ments, the polypeptide is a murine, chimeric, or
humanized antibody that specifically binds FOLRl. In certain embodiments, the polypeptide
having a certain tage of sequence identity to SEQ ID NOs:27-32 or 62-67 differs from
SEQ ID NOs:27-32 or 62-67 by conservative amino acid substitutions only.
Also provided are polypeptides comprising a variable light chain that is at
least about 85%, at least about 90%, at least about 95%, or at least about 99%, or is identical
to the variable light chain sequence of the antibody produced by the hybridoma having ATCC
deposit no. PTA-120196 or PTA-120197.
Also provided are polypeptides comprising a variable heavy chain that is at
least about 85%, at least about 90%, at least about 95%, or at least about 99%, or is identical
to the variable heavy chain sequence of the antibody produced by the hybridoma having
ATCC deposit no. PTA-120196 or 0197.
Also provided are antibodies and antigen-binding fragments thereof
comprising variable heavy and variable light chain ces that are at least about 85%, at
least about 90%, at least about 95%, or at least about 99%, or identical to the variable heavy
and variable light chain sequences of the antibody produced by the hybridoma having ATCC
deposit no. PTA-120196 or PTA-120197.
In certain embodiments, the antibody or antigen-binding nt is the
antibody produced by the hybridoma having ATCC deposit no. PTA-120196 or an antigen-
binding fragment thereof.
In certain embodiments, the antibody or antigen-binding nt is the
dy produced by the hybridoma having ATCC deposit no. PTA-120197 or an antigenbinding
fragment f.
Polypeptides can comprise one of the individual light chains or heavy chains
described herein. Antibodies and polypeptides can also comprise both a light chain and a
heavy chain. The light chain and heavy chain sequences of antibodies 2.1, 5.7, and 9.20 are
provided in Tables 5 and 6 below.
Table 5: ength heavy chain amino acid ces
Antibody Full-Length Heavy Chain Amino Acid Sequence (SEQ ID NO)
muFRIHC2-l QVQLQQSGPELVKPGASVRISCKASGYTFTNSYIHWVKKRPGQGLE
WIGWIYPESLNTQYNEKFKAKATLTADKSSSTSYMQLSSLTSEDSAV
("2'1")
YFCARRGIYYYSPYALDHWGQGASVTVSSAKTTPPSVYPLAPGSAA
QTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYT
LSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTV
PEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDV
EVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFP
APIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDIT
VEW0WNG OPAENYKNT I PIMNTNGSYFVYSKLNV 0KSNWEAGNT
LHEGLHNHHTEKSLSHSPGK SEQ ID NO:33
muFRIHC5-7 QVQLQQSGPEVVKPGASVRISCKASGYTFTNYYIHWVKQRPGQGLE
WIGWIYPGSFNVEYNEKFKAKATLTADKSSSTVYMQLSSLTSEDSA
("5' 7,,) VYFCARRGIYFYSPYALDYWGQGASVTVSSAKTTPPSVYPLAPGSA
AQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLY
TLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICT
VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDD
VEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAA
FPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPE
DITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAG
NTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:35)
muFRIHC9-20 QVQLQQSGPDLVKPGASVRISCKASGFTFTNYYIHWVKQRPGQGLE
WIGWIYPENVNVRYNDKFKAKATLTADKSSSTAYMQLSSLTSEDSA
("9 20")'
VYFCARRGIYYYSPYAMDYWGQGASVTVSSAKTTPPSVYPLAPGSA
AQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLY
TLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICT
VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDD
VEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAA
FPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPE
DITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAG
NTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:37)
muhuMOV19 QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSL
HPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDF
AVYYCTRYDGSRAMDYWGQGTTVTVSSASTKGPSVYPLAPGSAAQ
TNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLS
SSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPE
VSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEV
HTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAP
IEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVE
WQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFT
CSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:68)
Table 6: Full-length light chain amino acid sequences
Antibody Full-length Light Chain Amino Acid Sequence (SEQ ID NO)
muFRIHC2-1 SDVVLTQTPLSLPVNIGDQASISCKSSKSLLNSDGFTYLDWYLQKPG
QSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
("2.1")
PLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFL
DINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTL
TKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:34)
muFRIHC5-7 SDVVLTQTPLSLPVNIGDQASISCKSTESLLNSDGFTYLDWYLQKPG
QSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
("5 7")'
PLTFGGGTKLEVKRADAAPTVSIFPPSSEQLTSGGASVVCF
LNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT
LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID N036)
muFRIHC9-2O SDVVLTQTPLSLPVNLGDQASISCKSTKSLLNSDGFTYLDWYLQKP
GQSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYY
("9 20")'
CFQSNYLPLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCF
LNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT
LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID N03 8)
muhuMovl9 DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQ
PRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQ
SREYPYTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNN
FYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK
DEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:69)
Also provided are polypeptides that comprise: (a) a polypeptide having at least
about 90% sequence identity to SEQ ID , 35, or 37; and/or (b) a polypeptide having at
least about 90% sequence identity to SEQ ID NOs:34, 36, or 38. In certain embodiments, the
polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or at least about 99% sequence ty to SEQ ID NOs:33-
38. Thus, in certain embodiments, the polypeptide comprises (a) a polypeptide having at least
about 95% sequence identity to SEQ ID NOs:33, 35, or 37, and/or (b) a polypeptide having at
least about 95% sequence identity to SEQ ID NOs:34, 36, or 38. In certain ments, the
polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NOs:33,
, or 37; and/or (b) a polypeptide having the amino acid sequence of SEQ ID NOs:34, 36, or
38. In certain embodiments, the polypeptide is an antibody and/or the ptide
specifically binds FOLRl. In certain embodiments, the ptide is a murine, chimeric, or
humanized antibody that specifically binds FOLR]. In n embodiments, the polypeptide
having a certain percentage of sequence identity to SEQ ID NOs:33-38 s from SEQ ID
NOs:33-38 by conservative amino acid substitutions only.
Also provided are polypeptides comprising a light chain that is at least about
85%, at least about 90%, at least about 95%, or at least about 99%, or is cal to the light
chain sequence of the antibody produced by the oma having ATCC deposit no. PTA-
120196 or PTA-120197.
Also provided are polypeptides comprising a heavy chain that is at least about
85%, at least about 90%, at least about 95%, or at least about 99%, or is identical to the heavy
chain sequence of the antibody produced by the hybridoma having ATCC deposit no. PTA-
120196 or PTA-120197.
Also provided are antibodies and antigen-binding fragments thereof
comprising heavy and light chain sequences that are at least about 85%, at least about 90%, at
least about 95%, or at least about 99%, or identical to the heavy and light chain sequences of
the antibody produced by the hybridoma having ATCC deposit no. PTA-120196 or PTA-
120197.
The affinity or avidity of an antibody for an antigen can be determined
experimentally using any le method well known in the art, e.g., flow cytometry,
enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics
(e.g., BIACORETM analysis). Direct g assays as well as competitive binding assay
formats can be readily employed. (See, for example, Berzofsky, et al., "Antibody-Antigen
Interactions," In Fundamental logy, Paul, W. E., Ed, Raven Press: New York, NY.
(1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, NY. (1992);
and methods described herein. The measured affinity of a particular antibody-antigen
interaction can vary if measured under different conditions (e.g., salt concentration, pH,
temperature). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD
or Kd, K011, Koff) are made with standardized solutions of antibody and n, and a
standardized , as known in the art and such as the buffer described herein.
In one aspect, binding assays can be med using flow cytometry on cells
expressing the FOLRl antigen on the surface. For example, FOLRl-positve cells such as
SKOV3 can be incubated with varying concentrations of anti-FOLRI antibodies using 1 x105
cells per sample in 100 uL FACS buffer (RPMI—1640 medium supplemented with 2% normal
goat serum). Then, the cells can be pelleted, , and ted for 1 h with 100 uL of
FITC-conjugated nti-mouse or goat-anti—human IgG-antibody (such as is obtainable
from, for example Jackson Laboratory, 6 ug/mL in FACS buffer). The cells are then pelleted
again, washed with FACS buffer and resuspended in 200 uL of PBS containing 1%
formaldehyde. Samples can be ed, for example, using a FACSCalibur flow cytometer
with the HTS ell sampler and analyzed using CellQuest Pro (all from BD Biosciences,
San Diego, US). For each sample the mean fluorescence intensity for FL] (MFI) can be
exported and plotted t the antibody concentration in a semi-log plot to generate a
g curve. A sigmoidal dose—response curve is fitted for binding curves and ECSO values
are calculated using programs such as GraphPad Prism v4 with t parameters (GraphPad
software, San Diego, CA). ECSO values can be used as a measure for the apparent
dissociation constant "Kd" or "KD" for each antibody.
onal antibodies can be prepared using hybridoma methods, such as
those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma
method, a mouse, hamster, or other appropriate host animal, is immunized to elicit the
production by lymphocytes of antibodies that will specifically bind to an immunizing antigen.
Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are
isolated and fiised with a suitable myeloma cell line using, for e, polyethylene glycol,
to form hybridoma cells that can then be ed away from unfused lymphocytes and
myeloma cells. omas that produce monoclonal antibodies directed specifically against
a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro
binding assay (e.g., radioimmunoassay (RIA); -linked immunosorbent assay
(ELISA)) can then be propagated either in vitro culture using standard methods (Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites
tumors in an animal. The monoclonal antibodies can then be purified from the culture
medium or ascites fluid as described for polyclonal antibodies.
Alternatively monoclonal antibodies can also be made using recombinant
DNA s as described in US. Patent 4,816,567. The polynucleotides encoding a
monoclonal dy are isolated from mature B—cells or hybridoma cells, such as by RT-
PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy
and light chains of the antibody, and their sequence is determined using conventional
ures. The isolated polynucleotides encoding the heavy and light chains are then cloned
into suitable expression vectors, which when transfected into host cells such as E. coli cells,
simian COS cells, Chinese hamster ovary (CHO) cells, or a cells that do not
otherwise produce immunoglobulin protein, monoclonal dies are generated by the host
cells. Also, recombinant monoclonal antibodies or fragments thereof of the desired species
can be isolated from phage display libraries expressing CDRs of the desired species as
described (McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991, Nature,
352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).
The cleotide(s) encoding a monoclonal antibody can fiarther be
modified in a number of different manners using recombinant DNA logy to generate
alternative antibodies. In some ments, the constant domains of the light and heavy
chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions
of, for example, a human antibody to generate a chimeric antibody or 2) for a non-
immunoglobulin polypeptide to generate a fiasion antibody. In some embodiments, the
constant regions are truncated or removed to generate the desired antibody fragment of a
monoclonal antibody. Site-directed or high—density mutagenesis of the variable region can be
used to optimize specificity, affinity, etc. of a monoclonal antibody.
In some embodiments, the monoclonal antibody against the human FOLRl is
a zed antibody. In n embodiments, such antibodies are used therapeutically to
reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered
to a human subject.
Methods for engineering, humanizing or resurfacing non-human or human
antibodies can also be used and are well known in the art. A humanized, resurfaced or
similarly engineered antibody can have one or more amino acid es from a source that is
non-human, e.g., but not limited to, mouse, rat, rabbit, non-human primate or other mammal.
These man amino acid residues are replaced by residues that are often referred to as
"import" es, which are typically taken from an "import" variable, constant or other
domain of a known human sequence.
Such imported sequences can be used to reduce immunogenicity or reduce,
enhance or modify binding, affinity, on—rate, te, avidity, specificity, half-life, or any
other suitable characteristic, as known in the art. In general, the CDR es are directly
and most substantially involved in influencing FOLRl binding. Accordingly, part or all of the
non-human or human CDR sequences are maintained while the non-human sequences of the
variable and nt regions can be ed with human or other amino acids.
dies can also optionally be humanized, resurfaced, engineered or
human antibodies engineered with retention of high affinity for the n FOLRl and other
favorable biological properties. To achieve this goal, humanized (or human) or engineered
anti-FOLRl dies and resurfaced antibodies can be optionally prepared by a process of
analysis of the parental sequences and various conceptual humanized and ered
products using three-dimensional models of the parental, engineered, and humanized
sequences. Three-dimensional immunoglobulin models are commonly available and are
familiar to those skilled in the art. Computer programs are available which illustrate and
display probable three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of
es that influence the ability of the candidate immunoglobulin to bind its n, such
as FOLRl. In this way, framework (FR) es can be selected and combined from the
consensus and import sequences so that the desired antibody characteristic, such as sed
affinity for the target antigen(s), is achieved.
zation, resurfacing or engineering of antibodies of the present
invention can be performed using any known method, such as but not limited to those
described in, Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323
(1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296
; a and Lesk, J. M01. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci.
USA. 8924285 (1992); Presta et al., J. Immunol. 151:2623 (1993), US. Pat. Nos. 5,639,641,
,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;
,714,352; 6,204,023; 6,180,370; 5,693,762; 101; 5,585,089; 539; 4,816,567;
PCT/2 US98/16280; US96/18978; 9630; US91/05939; US94/01234; GB89/01334;
GB91/01134; GB92/01755; WO90/14443; WO90/14424; WO90/14430; EP 229246;
7,557,189; 7,538,195; and 7,342,110, each of which is ly incorporated herein by
reference, including the references cited therein.
In certain alternative embodiments, the antibody to FOLRl is a human
antibody. Human antibodies can be directly prepared using various techniques known in the
art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized
individual that produce an antibody directed against a target antigen can be generated (See,
e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);
Boemer et al., 1991, J. Immunol., 147 (1):86-95; and US. Patent 5,750,373). Also, the
human antibody can be selected from a phage library, where that phage library expresses
human antibodies, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-
314, Sheets et al., 1998, Proc. Nat’l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter,
1991, J. Mol. Biol., 2272381, and Marks et al., 1991, J. Mol. Biol., 222:581). Techniques for
the tion and use of antibody phage libraries are also described in US. Patent Nos.
,969,108, 6,172,197, 793, 6,521,404; 6,544,731; 313; 6,582,915; 6,593,081;
6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2007, J. Mol. Bio.,
doi:10.1016/j.jrnb.2007.12.018 (each of which is incorporated by reference in its entirety).
y maturation strategies and chain shuffling strategies (Marks et al., 1992,
Bio/Technology 102779-783, incorporated by reference in its entirety) are known in the art
and can be employed to generate high affinity human antibodies.
Humanized antibodies can also be made in transgenic mice containing human
immunoglobulin loci that are e upon immunization of producing the full oire of
human antibodies in the absence of nous immunoglobulin tion. This approach
is described in US. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
,661,016.
In certain embodiments are provided an antibody fragment to, for e,
increase tumor penetration. Various techniques are known for the production of dy
fragments. Traditionally, these fragments are derived via proteolytic digestion of intact
antibodies (for example Morimoto et al., 1993, Journal of Biochemical and Biophysical
Methods 242107-117; Brennan et al., 1985, Science, 229:81). In certain embodiments,
antibody fragments are produced inantly. Fab, Fv, and scFv antibody fragments can
all be expressed in and secreted from E. coli or other host cells, thus allowing the production
of large amounts of these fragments. Such antibody fragments can also be isolated from
antibody phage ies. The antibody fragment can also be linear antibodies as described in
US. Patent 5,641,870, for e, and can be monospecific or bispecific. Other techniques
for the production of antibody fragments will be apparent to the skilled tioner.
For the es of the present invention, it should be appreciated that
modified antibodies can comprise any type of variable region that provides for the association
of the antibody with the polypeptides of a human FOLRl. In this regard, the le region
can comprise or be derived from any type of mammal that can be induced to mount a
humoral response and generate immunoglobulins against the desired tumor associated
antigen. As such, the variable region of the modified antibodies can be, for example, of
human, murine, non-human primate (e.g., cynomolgus monkeys, macaques, etc.) or lupine
origin. In some embodiments both the variable and constant regions of the modified
immunoglobulins are human. In other embodiments the variable regions of compatible
antibodies (usually derived from a non-human source) can be engineered or specifically
ed to improve the binding properties or reduce the immunogenicity of the molecule. In
this respect, variable regions useful in the present invention can be humanized or otherwise
altered through the inclusion of imported amino acid ces.
In certain embodiments, the le domains in both the heavy and light
chains are altered by at least partial replacement of one or more CDRs and, if necessary, by
partial framework region replacement and sequence changing. Although the CDRs can be
derived from an antibody of the same class or even subclass as the antibody from which the
framework regions are derived, it is envisaged that the CDRs will be derived from an
antibody of different class and in certain embodiments from an antibody from a different
species. It may not be necessary to replace all of the CDRs with the complete CDRs from the
donor le region to transfer the antigen—binding capacity of one variable domain to
another. Rather, it may only be ary to transfer those residues that are necessary to
maintain the activity of the antigen-binding site. Given the explanations set forth in US. Pat.
Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the competence of those
skilled in the art, either by carrying out routine experimentation or by trial and error testing to
obtain a functional dy with reduced genicity.
Alterations to the le region notwithstanding, those skilled in the art will
appreciate that the modified antibodies of this invention will comprise antibodies (e. g., full-
length antibodies or immunoreactive fragments thereof) in which at least a fraction of one or
more of the constant region domains has been deleted or otherwise altered so as to provide
desired biochemical characteristics such as increased tumor localization or reduced serum
half-life when ed with an antibody of approximately the same immunogenicity
comprising a native or unaltered constant region. In some embodiments, the constant region
of the modified antibodies will se a human constant region. Modifications to the
constant region compatible with this ion comprise additions, deletions or substitutions
of one or more amino acids in one or more domains. That is, the modified antibodies
disclosed herein can comprise alterations or modifications to one or more of the three heavy
chain nt domains (CH1, CH2 or CH3) and/or to the light chain constant domain (CL).
In some embodiments, modified constant s wherein one or more domains are partially
or entirely d are contemplated. In some embodiments, the modified antibodies will
comprise domain deleted ucts or variants n the entire CH2 domain has been
removed (ACH2 constructs). In some embodiments, the omitted constant region domain will
be replaced by a short amino acid spacer (e.g., 10 residues) that provides some of the
molecular flexibility typically imparted by the absent constant region.
It will be noted that in certain embodiments, the modified antibodies can be
engineered to fuse the CH3 domain directly to the hinge region of the respective modified
antibodies. In other constructs it may be desirable to provide a peptide spacer between the
hinge region and the modified CH2 and/or CH3 domains. For example, compatible
constructs could be expressed wherein the CH2 domain has been deleted and the remaining
CH3 domain (modified or fied) is joined to the hinge region with a 5-20 amino acid
spacer. Such a spacer can be added, for instance, to ensure that the regulatory elements of the
nt domain remain free and accessible or that the hinge region remains flexible.
However, it should be noted that amino acid s can, in some cases, prove to be
immunogenic and elicit an unwanted immune response against the construct. Accordingly, in
certain ments, any spacer added to the construct will be relatively non-immunogenic,
or even omitted altogether, so as to maintain the desired biochemical qualities of the modified
antibodies.
Besides the deletion of whole constant region domains, it will be appreciated
that the antibodies of the present invention can be provided by the partial deletion or
substitution of a few or even a single amino acid. For example, the mutation of a single
amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc
binding and y increase tumor localization. Similarly, it may be desirable to simply
delete that part of one or more constant region domains that control the effector function
(e.g., complement ClQ binding) to be modulated. Such partial ons of the constant
regions can improve selected characteristics of the antibody (serum half-life) while leaving
other desirable ons associated with the subject constant region domain intact.
Moreover, as alluded to above, the constant s of the disclosed antibodies can be
modified through the mutation or substitution of one or more amino acids that enhances the
profile of the ing construct. In this respect it may be le to disrupt the activity
ed by a conserved binding site (e.g., Fc binding) while substantially maintaining the
configuration and immunogenic profile of the modified antibody. Certain embodiments can
comprise the addition of one or more amino acids to the constant region to enhance desirable
characteristics such as decreasing or increasing effector on or provide for more
cytotoxin or carbohydrate attachment. In such ments it can be ble to insert or
replicate specific sequences derived from ed nt region domains.
The present invention further embraces ts and equivalents which are
substantially homologous to the chimeric, humanized and human antibodies, or antibody
fragments thereof, set forth herein. These can contain, for example, conservative substitution
mutations, i.e., the substitution of one or more amino acids by similar amino acids. For
example, conservative substitution refers to the substitution of an amino acid with another
within the same general class such as, for example, one acidic amino acid with another acidic
amino acid, one basic amino acid with another basic amino acid or one neutral amino acid by
another neutral amino acid. What is intended by a conservative amino acid substitution is
well known in the art.
The polypeptides of the present invention can be recombinant polypeptides,
l polypeptides, or synthetic polypeptides comprising an antibody, or fragment thereof,
against a human FOLRl. It will be recognized in the art that some amino acid sequences of
the invention can be varied without significant effect of the structure or on of the
protein. Thus, the ion further includes variations of the polypeptides which show
substantial ty or which include regions of an antibody, or fragment thereof, against a
human folate receptor n. Such mutants include deletions, insertions, inversions,
repeats, and type substitutions.
The polypeptides and analogs can be further modified to contain onal
chemical moieties not normally part of the protein. Those derivatized moieties can improve
the solubility, the biological half life or absorption of the protein. The moieties can also
reduce or eliminate any ble side effects of the proteins and the like. An overview for
those moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed.,
Mack Publishing Co., Easton, PA (2000).
The isolated polypeptides described herein can be produced by any suitable
method known in the art. Such methods range from direct protein tic s to
constructing a DNA sequence encoding isolated polypeptide sequences and expressing those
sequences in a suitable transformed host. In some embodiments, a DNA sequence is
constructed using recombinant technology by isolating or synthesizing a DNA sequence
encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by
pecific mutagenesis to e functional analogs f. See, e.g., Zoeller et al., Proc.
Nat’l. Acad. Sci. USA 81 :5662-5066 (1984) and US Pat. No. 4,588,585.
In some embodiments a DNA sequence encoding a polypeptide of interest
would be constructed by chemical synthesis using an oligonucleotide synthesizer. Such
ucleotides can be designed based on the amino acid sequence of the desired
polypeptide and selecting those codons that are favored in the host cell in which the
recombinant polypeptide of interest will be produced. Standard methods can be applied to
synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest.
For e, a complete amino acid sequence can be used to construct a back-translated
gene. Further, a DNA oligomer containing a nucleotide sequence coding for the ular
ed polypeptide can be synthesized. For e, several small oligonucleotides coding
for portions of the desired polypeptide can be synthesized and then ligated. The individual
oligonucleotides typically contain 5' or 3' ngs for complementary assembly.
Once assembled (by synthesis, site-directed mutagenesis or another method),
the polynucleotide sequences encoding a ular isolated polypeptide of interest will be
inserted into an sion vector and operatively linked to an expression control sequence
appropriate for expression of the protein in a desired host. Proper assembly can be confirmed
by tide sequencing, restriction mapping, and expression of a biologically active
polypeptide in a suitable host. As is well known in the art, in order to obtain high expression
levels of a transfected gene in a host, the gene must be operatively linked to transcriptional
and ational expression control sequences that are functional in the chosen sion
host.
In certain embodiments, recombinant expression vectors are used to amplify
and express DNA encoding antibodies, or fragments thereof, against human FOLRl.
Recombinant expression vectors are replicable DNA constructs which have synthetic or
cDNA-derived DNA fragments encoding a polypeptide chain of an OLRl antibody, or
fragment thereof, operatively linked to suitable transcriptional or translational regulatory
elements derived from ian, microbial, viral or insect genes. A transcriptional unit
generally comprises an assembly of (1) a genetic element or elements having a regulatory
role in gene sion, for example, riptional promoters or ers, (2) a structural
or coding sequence which is transcribed into mRNA and translated into protein, and (3)
appropriate ription and translation initiation and termination sequences. Such
tory ts can include an operator sequence to control transcription. The ability to
replicate in a host, usually conferred by an origin of replication, and a selection gene to
facilitate recognition of transformants can onally be incorporated. DNA regions are
operatively linked when they are functionally related to each other. For example, DNA for a
signal peptide (secretory ) is operatively linked to DNA for a polypeptide if it is
expressed as a precursor which participates in the secretion of the polypeptide; a promoter is
operatively linked to a coding ce if it controls the transcription of the sequence; or a
ribosome binding site is operatively linked to a coding sequence if it is positioned so as to
permit translation. Structural elements intended for use in yeast expression systems include a
leader sequence ng ellular secretion of translated protein by a host cell.
Alternatively, where recombinant protein is expressed without a leader or transport sequence,
it can include an N-terminal methionine residue. This residue can optionally be subsequently
cleaved from the expressed recombinant protein to provide a final product.
The choice of expression control sequence and expression vector will depend
upon the choice of host. A wide variety of expression host/vector combinations can be
employed. Useful expression vectors for eukaryotic hosts, e, for example, vectors
comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and
cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial
plasmids, such as plasmids from Escherichia coli, including pCR l, pBR322, pMB9 and their
derivatives, wider host range plasmids, such as M13 and filamentous single-stranded DNA
phages.
Suitable host cells for expression of a binding polypeptide or antibody
(or a FOLRl protein to use as an antigen) include prokaryotes, yeast, insect or higher
eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram
negative or gram positive organisms, for example E. 0012' or bacilli. Higher eukaryotic cells
e established cell lines of mammalian origin. ree translation systems could also
be employed. Appropriate cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described by s et a1. ng Vectors: A
Laboratory Manual, Elsevier, N.Y., 1985), the relevant disclosure of which is hereby
incorporated by reference. Additional information regarding methods of n production,
including antibody production, can be found, e.g., in US. Patent Publication No.
2008/0187954, US. Patent Nos. 6,413,746 and 6,660,501, and International Patent
Publication No. WO 23, each of which is hereby incorporated by reference herein in
its ty.
Various mammalian or insect cell culture systems are also advantageously
employed to express recombinant protein. Expression of recombinant proteins in mammalian
cells can be performed because such proteins are generally correctly folded, appropriately
modified and completely fiinctional. Examples of suitable mammalian host cell lines include
HEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells, described by Gluzman
(Cell 23:175, 1981), and other cell lines including, for example, L cells, C127, 3T3, Chinese
hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors can
comprise nontranscribed elements such as an origin of replication, a suitable er and
enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed
sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites, and transcriptional ation
sequences. Baculovirus systems for production of heterologous proteins in insect cells are
reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).
The ns produced by a transformed host can be purified according to any
suitable method. Such standard methods include tography (e.g., ion exchange, affinity
and sizing column chromatography), centrifugation, differential solubility, or by any other
standard technique for protein purification. y tags such as hexahistidine, maltose
binding domain, influenza coat ce and glutathione—S-transferase can be attached to the
n to allow easy purification by passage over an appropriate affinity column. Isolated
proteins can also be physically terized using such techniques as proteolysis, nuclear
ic resonance and x-ray crystallography.
For example, supematants from systems which e recombinant n
into culture media can be first concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to a suitable purification
matrix. Alternatively, an anion exchange resin can be ed, for example, a matrix or
ate having pendant diethylaminoethyl (DEAE) groups. The matrices can be
acrylamide, agarose, dextran, cellulose or other types commonly employed in protein
purification. Alternatively, a cation exchange step can be employed. Suitable cation
exchangers include various insoluble es comprising sulfopropyl or carboxymethyl
groups. y, one or more reversed-phase high performance liquid tography (RP-
HPLC) steps employing hydrophobic RP—HPLC media, e.g., silica gel having pendant methyl
or other aliphatic groups, can be ed to r purify a FOLRl-binding agent. Some
or all of the foregoing purification steps, in s combinations, can also be employed to
provide a homogeneous recombinant protein.
Recombinant protein produced in bacterial culture can be isolated, for
e, by initial extraction from cell pellets, followed by one or more concentration,
salting-out, aqueous ion exchange or size exclusion chromatography steps. High performance
liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells
employed in expression of a recombinant protein can be disrupted by any ient method,
including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
Methods known in the art for purifying antibodies and other proteins also
include, for example, those bed in US. Patent Publication Nos. 2008/0312425,
2008/0177048, and 2009/0187005, each of which is hereby incorporated by reference herein
in its entirety.
III. Polynucleotides
In certain embodiments, the invention encompasses polynucleotides
comprising cleotides that encode a polypeptide that cally binds a human FOLRl
receptor or a fragment of such a polypeptide. For example, the invention provides a
polynucleotide comprising a nucleic acid sequence that encodes an antibody to a human
FOLRl or encodes a fragment of such an antibody. The polynucleotides of the invention can
be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and
synthetic DNA; and can be double-stranded or single—stranded, and if single stranded can be
the coding strand or non-coding sense) strand. In some embodiments, the
polynucleotide is a cDNA or a DNA lacking one more endogenous s.
In some embodiments, a polynucleotide is a non-naturally occurring
polynucleotide. In some embodiments, a polynucleotide is recombinantly ed.
In certain embodiments, the polynucleotides are isolated. In certain
embodiments, the polynucleotides are substantially pure. In some embodiments, a
polynucleotide is purified from natural components.
The invention provides a polynucleotide comprising a polynucleotide
encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID
NOs:3-38 and 59-67. Also provided is a polynucleotide encoding a ptide having at
least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about
99% sequence identity to SEQ ID 38 and 59—67.
The invention further provides a polynucleotide comprising a sequence
selected from those shown in Tables 7 and 8 below.
Table 7: le heavy chain polynucleotide sequences
Antibody Variable Heavy Chain Polynucleotide Sequence (SEQ ID NO)
muFRIHC2-l caggtccaactgcagcagtctggacctgagctggtgaagcctggggcttcagtgaggatatcctgcaagg
cttctggctacaccttcacaaactcctatattcactgggtgaaaaagaggcctggacagggacttgagtgga
("2 l")'
ttggatggatttatcctgaaagtcttaatactcaatacaatgagaagttcaaggccaaggccacactgactgct
gacaagtcctccagcacatcctacatgcagctcagcagtctgacctctgaggactctgcggtctatttctgtg
caagaaggggtatttattactactctccctatgctctggaccactggggtcaaggagcctcagtcaccgtctc
ctca (SEQ ID NO:39)
muFRIHC5-7 caactgcagcagtctggacctgaggtggtgaagcctggggcttcagtgaggatatcctgcaagg
cttctggctacaccttcacaaactactatatacactgggtgaagcagaggcctggacagggacttgagtgga
("5 7")'
ttggatggatttatcctggaagttttaatgttgagtacaatgagaagttcaaggccaaggccacactgactgc
agacaaatcctccagcacagtctacatgcaactcagcagcctgacctctgaggactctgcggtctatttctgt
aggggtatttatttctactctccctatgctttggactactggggtcaaggagcctcagtcaccgtctc
ctca (SEQ ID NO:41)
muFRIHC9-20 caggtccaactgcagcagtctggacctgacctggtgaagcctggggcttcagtgaggatatcctgcaagg
cttctggcttcaccttcacaaactactatatacactgggtgaagcagaggcctggacagggacttgagtgga
("9.20")
ttggatggatttatcctgaaaatgttaatgttaggtacaatgacaagttcaaggccaaggccacactgactgc
agacaaatcctccagcacagcctacatgcagctcagcagcctgacctctgaggactctgcggtctatttctg
tgcaagaaggggtatttattactactctccctatgctatggactactggggtcaaggagcctcagtcaccgtct
cctca (SEQ ID NO:43)
huFRIHCZ-l aagcttgccaccATGGGTTGGAGCTGCATTATCCTTTTCCTTGTGGCTA
CTGGCGTTCACTCTCAGGTACAATTGGTTCAGTCAGGA
(resurfaced)
GCCGAGGTCGTAAAGCCCGGTGCCAGTGTGAAGATCTCATGCAA
GGCAAGCGGTTATACTTTTACAAACTCTTACATTCATTGGGTGAA
AAAGCGGCCCGGCCAGGGTCTCGAATGGATCGGCTGGATCTACC
CAGAAAGTCTGAACACTCAATACAACCAGAAGTTTCAGGGTAAG
GCAACTCTCACTGCCGACAAGAGCTCTAGCACAAGCTATATGCA
GTTGTCTAGTTTGACAAGCGAGGATAGCGCAGTTTACTTTTGTGC
TCGGCGTGGTATTTATTACTACTCACCTTATGCTCTGGATCACTG
GGGACAGGGTGCCTCTGTTACCGTTTCCAGTGCATCCACCaagggcc
c (SEQ ID NO:70)
huFRIHCZ-l aagcttgccaccATGGGCTGGAGCTGCATAATCCTCTTCCTCGTAGC
CACTGGGGTGCATTCTCAAGTACAGTTGGTGCAGTC
(grafted)
CGGAGCTGAAGTCAAGAAGCCAGGGGCTTCTGTTAAGGTGA
GCTGTAAGGCTTCCGGATATACCTTCACAAACAGTTATATCC
ATTGGGTGAGGCAAGCTCCAGGCCAGGGTCTCGAATGGATG
GGATGGATCTACCCCGAGAGTCTGAACACCCAGTACAACGA
GAAGTTCAAGGCACGTGTGACCATGACAAGAGACACCTCCA
TCAGTACAGCCTATATGGAATTGAGCCGTCTCAGAAGTGATG
ATACAGCAGTGTACTACTGCGCCAGGCGGGGCATCTACTACT
ACAGCCCATACGCTCTCGACCACTGGGGACAAGGAACACTG
GTAACCGTAAGCTCAGCTTCTACAaagggccc (SEQ ID NO:7l)
Table 8: le light chain polynucleotide sequences
Antibody Variable Light Chain Polynucleotide Sequence (SEQ ID NO)
muFRIHCZ- l agtgatgttgttctgacccaaactccactctctctgcctgtcaatattggagatcaagcctctatctcttgcaagt
cttctaagagtcttctgaatagtgatggattcacttatttggactggtacctgcagaagccaggccagtctcca
("2 l")'
cagctcctaatatatttggtttctaatcatttttctggagttccagacaggttcagtggcagtgggtcaggaaca
acactcaagatcagcagagtggaggctgaggatttgggagtttattattgcttccagagtaactatctt
cctctcacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO:40)
muFRIHC5-7 gttgttctgacccaaactccactctctctgcctgtcaatattggagatcaagcctctatctcttgcaagt
ctactgagagtcttctgaatagtgatggattcacttatttggactggtacctgcagaagccaggccagtctcca
("5 7")‘
cagctcctaatatatttggtttctaatcatttttctggagttccagacaggttcagtggcagtgggtcaggaaca
gatttcacactcaagatcagcagagtggaggctgaggatttgggagtttattattgcttccagagtaactatctt
cctctcacgttcggaggggggaccaagctggaagtaaaacgg (SEQ ID NO:42)
muFRIHC9-20 agtgatgttgttctgacccaaactccactctctctgcctgtcaatcttggagatcaagcctctatctcttgcaagt
ctactaagagtcttctgaatagtgatggattcacttatttggactggtacctgcagaagccaggccagtctcca
("9.20")
cagctcctaatatatttggtttctaatcatttttctggagttccagacaggttcagtggcagtgggtcaggaaca
gatttcaccctcaagatcagcagagtggaggctgaggatttgggagtttattattgcttccagagtaactatctt
cctctcacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO:44)
huFRIHCZ-l V. gaattcgccaccATGGGTTGGTCATGTATAATACTTTTCCTGGTAGC
TACTGCTACTGGTGTGCATTCAGATGTGGTGCTGACTCAGTC
1.0 (resurfaced)
ACCCTTGTCTCTCCCAGTCAATCTTGGGCAGCCAGCATCTATC
AGCTGCCGAAGCAGCAGGTCTCTCCTGAACTCCGATGGCTTT
ACTTATCTTGACTGGTATCTCCAGAAGCCAGGACAGTCCCCC
CGGCTGCTCATCTACCTGGTTTCTAATCATTTTAGTGGCGTCC
CTGACCGCTTCTCTGGGAGTGGAAGTGGGACCGATTTTACAC
TGAAGATCTCCAGGGTCGAAGCTGAGGACCTTGGGGTTTACT
ACTGTTTCCAGAGCAACTACCTTCCCTTGACATTCGGCCAGG
AGCTGGAAATCAAGcgtacg (SEQ ID NO:72)
huFRIHCZ-l V. gaattcgccaccATGGGTTGGTCTTGTATCATTCTGTTCCTGGTCGC
CACTGCCACAGGAGTTCACTCAGACGTGGTACTCACACAATC
1.01 (resurfaced)
TCCCCTTTCCCTGCCTGTGAACCTGGGACAGCCAGCCTCAAT
CAGTTGCAAGAGCTCTAAATCTCTGCTCAATAGCGATGGCTT
TACCTACTTGGATTGGTACCTCCAGAAGCCCGGCCAGTCTCC
TCGGCTCCTGATTTACCTTGTTTCAAATCACTTTTCAGGCGTG
CCTGACCGGTTCTCCGGATCTGGCTCAGGGACAGACTTCACC
CTGAAGATCTCCCGCGTCGAGGCAGAGGATCTCGGCGTGTAT
TACTGTTTCCAAAGTAACTACCTGCCATTGACTTTTGGACAA
GGAACTAAACTGGAAATCAAAcgtacg (SEQ ID NO:73)
huFRIHCZ-l V. gaattcgccaccATGGGATGGAGTTGTATTATTCTGTTCTTGGTCGC
TACTGCAACAGGCGTTCATTCTGACATCGTAATGACCCAGAC
1.0 (grafted)
ACCTCTGAGTCTGAGTGTCACTCCCGGCCAGCCCGCCTCTATT
TCATGTCGTAGCTCTCGCTCCCTGCTCAATTCCGACGGTTTTA
CCTACTTGGACTGGTATCTTCAGAAACCTGGGCAGAGCCCTC
AGCTTCTGATCTATCTGGTGTCCAATCACTTCAGTGGCGTCCC
AGACCGATTTTCCGGAAGCGGAAGCGGAACCGACTTTACCCT
GAAGATATCCCGCGTCGAAGCAGAGGACGTGGGCGTGTATT
ATTGCTTTCAAAGCAATTACTTGCCATTGACTTTCGGACAAG
GCACAAAACTGGAGATTAAGcgtacg (SEQ ID NO:74)
huFRIHCZ-l V. gaattcgccaccATGGGCTGGTCATGCATCATACTGTTCCTGGTGGC
TACAGCAACCGGGGTGCACAGCGATATTGTTATGACACAGAC
1.01 (grafted)
GAGTTTGTCAGTGACCCCCGGCCAGCCAGCCTCTAT
CAAGTCCTCAAAAAGTCTCCTGAATAGCGATGGCTT
TACCTACCTCGACTGGTATCTTCAGAAGCCCGGTCAAAGCCC
TCAGCTGCTGATATATCTGGTGTCTAACCATTTTAGCGGAGTC
CCCGACCGCTTTTCAGGCTCCGGCAGTGGCACCGACTTCACC
CTTAAGATTTCTCGCGTGGAGGCTGAAGATGTAGGGGTCTAC
TACTGTTTCCAGTCAAACTACCTGCCACTGACCTTTGGTCAAG
GCACTAAGCTCGAAATTAAGcgtacg (SEQ ID NO:75)
Also provided is a polynucleotide having at least about 95%, at least about
96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any
one of SEQ ID -44.
Also provided are cleotides encoding a variable light chain that is at
least about 85%, at least about 90%, at least about 95%, or at least about 99%, or is identical
to the variable light chain sequence of the antibody produced by the hybridoma having ATCC
deposit no. PTA-120196 or PTA-120197.
Also provided are polynucleotides comprising a variable light chain-encoding
sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about
99%, or is identical to the variable light chain-encoding sequence that encodes the variable
light chain of the antibody produced by the hybridoma having ATCC deposit no. PTA-
120196 or PTA-120197.
Also provided are polynucleotides encoding a variable heavy chain that is at
least about 85%, at least about 90%, at least about 95%, or at least about 99%, or is identical
to the variable heavy chain ce of the antibody produced by the hybridoma having
ATCC deposit no. PTA—120196 or PTA—120197.
Also provided are polynucleotides comprising a variable heavy chain-
encoding sequence that is at least about 85%, at least about 90%, at least about 95%, or at
least about 99%, or is identical to the variable heavy encoding sequence that encodes
the le heavy chain of the antibody produced by the oma having ATCC deposit
no. PTA-120196 or PTA-120197.
In certain embodiments the polynucleotides comprise the coding sequence for
the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for
example, in expression and secretion of a polypeptide from a host cell (e. g., a leader sequence
which functions as a secretory sequence for controlling transport of a polypeptide from the
cell). The polypeptide having a leader sequence is a preprotein and can have the leader
sequence cleaved by the host cell to form the mature form of the polypeptide. The
polynucleotides can also encode for a proprotein which is the mature protein plus additional
' amino acid residues. A mature protein having a prosequence is a proprotein and is an
inactive form of the n. Once the prosequence is cleaved an active mature protein
remains.
In n embodiments the polynucleotides comprise the coding sequence for
the mature polypeptide fused in the same g frame to a marker sequence that allows, for
e, for purification of the encoded polypeptide. For example, the marker sequence can
be a hexa-histidine tag ed by a pQE—9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a ial host, or the marker ce can be
a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a
mammalian host (e. g., COS-7 cells) is used.
The present ion fiirther relates to variants of the hereinabove described
polynucleotides encoding, for example, nts, analogs, and derivatives.
The polynucleotide variants can contain alterations in the coding regions, non-
coding regions, or both. In some ments the polynucleotide variants contain alterations
which produce silent substitutions, additions, or deletions, but do not alter the properties or
activities of the encoded polypeptide. In some embodiments, nucleotide variants are
produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide
variants can be produced for a variety of reasons, e.g., to optimize codon sion for a
particular host (change codons in the human mRNA to those preferred by a ial host
such as E. 6011').
s and cells comprising the polynucleotides described herein are also
provided.
IV. Biological samples
Biological samples are often fixed with a fixative. Aldehyde fixatives such as
formalin (formaldehyde) and glutaraldehyde are lly used. Tissue samples fixed using
other fixation techniques such as alcohol immersion fora and Kopinski, J. hem.
Cytochem. (1986) 34:1095) are also suitable. The samples used may also be embedded in
paraffin. In one embodiment, the 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 Jan;125(1):107—13).
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 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. The ical sample can also be fluidic extracts,
blood, plasma, serum, spinal fluid, lymph fluid, and or c preparations.
V. Correlation of FOLRl sion and therapeutic y
The antibody maytansinoid conjugate (AMC) IMGN853 comprises the
FOLRl-binding monoclonal antibody, huMovl9 (M9346A), conjugated to the maytansinoid,
DM4 (N(2')-deacetyl-N2'-(4-rnercapto-4—methyloxopentyl)-n1aytansine), attached Via the
cleavable sulfo-SPDB (N-succinirnidyl 4—(2—pyridyldithio)-2—sulfobutanoate) linker. The
dy sequences of IMGN853 (huMovl9) are provided below as SEQ ID NOs: 45 and 47,
and IMGN853 and huMovl9 are described in US Appl. Pub. No. 2012/0009181 (now
8,557,966), which is herein incorporated by reference in its entirety.
SEQ ID NO:45 — huMovl9 VHC
SGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDG
KFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQG
TTVTVSS
SEQ ID NO:46 - huMovl9 VLCVl.00
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNL
EAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR
SEQ ID NO:47 - huMovl9 VLCVl.60
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNL
EAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR
SEQ ID NO:48 - huMovl9 VLC CDRl
KASQSVSFAGTSLMH
SEQ ID NO:49 - huMovl9 VLC CDRZ
RASNLEA
SEQ ID NO:50 - huMovl9 VLC CDR3
QQSREYPYT
SEQ ID NO:51 - huMovl9 VHC CDRl
GYFMN
SEQ ID NO:52 - huMovl9 VHC CDR2 — Kabat Defined
RIHPYDGDTFYNQKFQG
SEQ ID NO:53 — huMovl9 VHC CDR2 — Abm Defined
SEQ ID NO:54 - huMovl9 VHC CDR3
YDGSRAMDY
SEQ ID NO:55 - huMovl9 HC amino acid sequence
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDG
DTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQG
TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:56 - huMovl9 0
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNL
EAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAA
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:57 - huMovl9 LCvl.60
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNL
EAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAA
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:58 — muMovl9 VHC CDR2 — Kabat Defined
RIHPYDGDTFYNQNFKD
IMGN853 is currently in clinical development for various therapeutic
indications which include FOLRl positive ovarian cancer, non-small cell lung ,
endometrioid cancer, renal cancer, and other epithelial malignancies. Ovarian cancers exhibit
the greatest FOLRl ance and are considered the major tions for treatment with
3 (Antony AC. Ann Rev Nutr 16:501—21 (1996); Yuan Y et a1. Hum Pathol
40(10):l453-l460 (2009)). Measuring levels of FOLR] in patient plasma samples can help
identify patient populations more likely to respond to AMC treatment.
In certain embodiments, the invention provides a method for fying
subjects that are likely to respond favorably to FOLRl-targeting anti-cancer therapies due to
elevated expression levels of FOLRl being expressed in the subject, in particular using
antibodies and antigen-binding fragments f provided herein that can detect a dynamic
range of FOLRl expression levels, e. g., in IHC.
Evaluation of patient samples and correlation to in viva efficacy using
aft models demonstrates the power of the expression analysis for selecting subjects
more likely to respond to treatment. IHC provides a score for FOLRl expression on tumor
cells: 0 (no expression) to 3 (or 3+) (very high levels of expression). In vivo data using
xenograft models demonstrates that samples scoring 2, or 3 (or 3+) for FOLRl expression
have an increased likelihood to d to FOLRl-targeted anti—cancer therapies at clinically-
relevant doses of FOLRl conjugates (see e.g., US. Provisional Application Nos.
61/823,317 and 61/828,586 and International Application No. , all of
which are herein incorporated by reference in their entireties). Thus, identification of
individuals having an elevated FOLRl score would help identify those individuals who might
d to a ally relevant dosage. Moreover, expression of more uniform levels of
FOLRl provides better correlation with therapeutic benefit. Thus, a homogenous staining
uniformity or a combination of increased staining with heterogenous staining uniformity can
te increased FOLRl expression. For example, scores of greater than 2 hetero may be
used as a patient selection criterion for treatment with a FOLRl therapeutic agent (see e.g.,
U.S. Published Application No. 2012/0282175, which is herein incorporated by nce in
its entirety).
FOLRl expression analysis also identifies patients in whom decreased levels
of a FOLRl-targeting anti-cancer therapy (“low dose therapy”) can be effective to cause anti-
tumor responses. As is iated in the art, compounds are generally administered at the
st dosage that achieves the desired therapeutic response. This is specifically important
for eutics that cause clinical side effects. The ability to recognize those subjects with
ed FOLRl expression levels allows for minimization of the dosage of the FOLRltargeting
eutic, thus decreasing possible side effects, while maintaining therapeutic
efficacy.
Accordingly, the antibodies and antigen-binding nts provided herein
are particularly advantageous for use in such methods because they are capable of detecting a
dynamic range of FOLRl expression levels, e.g., in IHC.
VI. Shed antigen assay
Measuring levels of circulating n in patient plasma s (shed
antigen) can help identify patient populations more likely to respond to treatment, e.g.,
antibody maytansinoid conjugate (AMC) treatment. High levels of shed antigen have been
reported to markedly affect the pharmacokinetics of therapeutic antibodies (Tolcher A. et al.
20th Symposium on Molecular Targets and Cancer Therapeutics; October 21-24, 2008;
Geneva, Switzerland: EORTC-NCI-AACR, p163, #514; Baselga J, et al. J Clin Oncol
14:737-744 (1996)). It is likely that shed n levels from patient plasma samples will be
variable depending on s such as n , disease tions, and disease course.
Currently shed antigen levels in disease indications for the anti-FOLRl immunoconjugate
IMGN853 have been insufficiently examined while correlation with solid tumor expression is
limited. While elevation of FOLRI has been reported in ovarian arcinomas, data
suggests that it is not elevated in other FOLR1+ tumor tions, such as small cell lung
carcinoma (Mantovani LT, et al. Eur J Cancer 30A(3):363-9 (1994); Basal E, et al. PLoS
ONE 4(7): e6292 (2009)). The present method allows for detection of the FOLRI receptor in
the presence of high folic acid using the antibodies and antigen-binding fragments thereof
that are provided herein and are capable of detecting dynamic ranges of shed FOLRl.
Previous assays have used Movl9 in the design of the assay. Since IMGN853 contains
MOV19 and in one embodiment is the targeted therapy of the invention, it is Vital that the
method detects FOLRl in the presence or absence of M0V19. Previous assays that use
MOV19 have competitive effects and will detect significantly less or no FOLRl in patients
receiving IMGN853 treatment.
In one embodiment, the present method for detecting FOLRl in human
sourced fluid samples uses a traditional sandwich ELISA format. In one embodiment, the
method uses a capture agent (i.e., antibody) to FOLRl attached to a solid support. In one
ment, the solid support is a microtiter plate. To this, the sample es fluids,
plasma, etc.) is added without dilution, and is detected by a different detection agent (a
different antibody), which does not ere with the binding of the first capture agent. The
detection agent is then detected through the use of a secondary detection agent (biotin /
streptavidin, anti-human secondary mono or polyclonal antibody, etc.) which can bind more
than one time to the first detection agent, thus amplifying the signal of detection. The
secondary detection agent is then quantified by the use of some other means (e.g.,
TMB/peroxidase, scintillation counting, fluorescent probes, etc.). Additionally, the assay
detects FOLRI and is not negatively impacted by the presence of Mov19, IMGN853, other
FOLRl family members, or folic acid.
The assays of the present ion include assays both to select ts
eligible to receive FOLRl-based therapy and assays to monitor patient response. Assays for
response prediction are run before y selection, and levels of FOLRl may impact
therapy decisions. For monitoring patient response, the assay is run at the tion of therapy
to establish baseline (or predetermined) levels of FOLRl in the sample. The same sample is
then d and the levels of FOLRl compared to the baseline or predetermined levels. As
used herein, the term "predetermined level" refers lly to an assay cutoff value that is
used to assess diagnostic results by comparing the assay results against the predetermined
level, and where the predetermined level already has been linked or associated with various
clinical parameters (e.g., monitoring whether a subject being treated with a drug has achieved
an efficacious blood level of the drug, monitoring the response of a subject receiving
treatment for cancer with an anti-cancer drug, ring the response of a tumor in a t
receiving treatment for said tumor, etc.). The predetermined level may be either an absolute
value or a value normalized by subtracting the value obtained from a patient prior to the
initiation of therapy. An example of a predetermined level that can be used is a baseline level
obtained from one or more subjects that may optionally be suffering from one or more
diseases or conditions. The comparison (or informational analysis) of the level of the assayed
biomarker with the baseline or predetermined level can be done by an automated ,
such as a software program or igence system that is part of, or compatible with, the
equipment (e.g., computer platform) on which the assay is carried out. Alternatively, this
comparison or informational is can be done by a physician. In one embodiment, where
the levels remain the same or decrease, the therapy may be effective and can be continued.
Where significant increase over baseline level (or ermined level) occurs, the patient
may not be responding. In another embodiment, an increase in shed FOLRl levels may be
indicative of increased cell death and increased release of the shed FOLRl. In this
embodiment, an increase in shed FOLRl is indicative of therapeutic efficacy.
The assays of the present invention can be performed by any protein assay
methods. Protein assay methods useful in the invention are well known in the art and include
immunoassay methods involving binding of a specific unlabeled or labeled antibody or
protein to the expressed protein or fragment of FOLR]. Usefiil immunoassay s
include both solution phase assays conducted using any format known in the art, such as, but
not limited to, Biacore, time resolved fluorescence energy er (TR-FRET), an ELISA
format, ich, forward and e competitive inhibition) or a cence polarization
format, and solid phase assays such as immunohistochemistry. The FOLRl antibodies and
antigen-binding fragments f ed herein are particularly useful for these
immunoassay methods because, for example, they are able to detect a dynamic range of
FOLRl.
VII. Circulating Tumor Cell Assays
The anti-FOLRl antibodies described herein can also be used for the detection
of FOLRl in a ating tumor cell assay. Circulating tumor cells (CTCs) are cells that
have shed into the vasculature from a tumor and circulate in the bloodstream. CTCs are
present in circulation in extremely low numbers. In general, CTCs are enriched from patient
blood or plasma by s techniques known in the art. CTCs can be stained for specific
s using methods known in the art including, but not limited to, flow try-based
methods and IHC-based methods. CTCs may be stained for protein s unique to the
tumor cells, which allows for the identification and distinction of CTCs from normal blood
cells. CTCs can also be stained for FOLRl using the antibodies provided herein including
but not limited to 2.1, 5.7, and 9.20. CTC analysis can also include quantitative analysis of
the number of CTCs and/or the number of FOLRl positive CTCs. The FOLRl antibodies
described herein can be used to stain the CTCs isolated from a subject having a cancer to
e the FOLRl present in the CTCs. An increase in FOLRl expressing CTCs can help
identify the subject as having a cancer that is likely to respond to FOLRl based therapy or
allow for optimization of a therapeutic n with a FOLRl antibody or immunoconjugate.
CTC FOLRl quantitation can provide information on the stage of tumor, response to therapy
and/or disease progression. It can be used as prognostic, predictive or pharmacodimamic
biomarker. In addition, staining of CTCs for FOLRl using the antibodies provided herein,
can be used as a liquid biopsy either alone or in combination with onal tumor marker
analysis of solid biopsy samples.
VIII. Detection
The present invention further provides antibodies against FOLRl, generally of
the monoclonal type, that are linked to at least one agent to form a detection dy
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 le or moiety.
Such a le 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,
radiolabels, haptens, fluorescent , phosphorescent molecules, chemiluminescent
molecules, chromophores, luminescent les, photoaffinity molecules, colored particles
and/or ligands, such as biotin.
n examples of antibody conjugates are those conjugates in which the
antibody or antigen-binding fragment thereof provided herein 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 or
antigen-binding fragment to which they are attached to be detected, and/or r quantified
if desired.
Many appropriate imaging agents are known in the art, as are methods for
their attachment to dies (see, e.g., US. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509,
each incorporated herein by reference). The g moieties used can be paramagnetic ions;
radioactive isotopes; fluorochromes; NMR-detectable substances; and/or X-ray imaging, for
example.
Exemplary fluorescent labels contemplated for use as binding agent (e.g.,
antibody) conjugates include Alexa 350, Alexa 430, Alexa 488, AMCA, BODIPY 630/650,
BODIPY 650/665, -FL, BODIPY-R6G, BODIPY-TMR, -TRX, Cascade
Blue, Cy3, Cy5,6-FAM, Dylight 488, Fluorescein Isothiocyanate (FITC), Green fluorescent
protein (GFP), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514,
Pacific Blue, Phycoerythrin, REG, Rhodamine Green, Rhodamine Red, tetramethyl in
(TMR) Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, Texas Red, and
derivatives of these labels (i.e., nated analogues, modified with isothiocynate or other
linker for conjugating, etc.), for example. An exemplary abel is tritium.
Antibody or antigen-binding fragment detection conjugates contemplated in
the present invention include those for use in vitro, where the antibody or fragment is linked
to a secondary g ligand and/or to an enzyme (an enzyme tag) that will te a
d product upon t with a chromogenic substrate. The FOLR1 antibodies and
antigen-binding fragments thereof provided herein are particularly useful for conjugates
methods because, for e, they are able to detect a dynamic range of FOLR1. Examples
of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase
and/or glucose oxidase. In some embodiments, 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 US. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
Molecules containing azido groups may also be used to form covalent bonds
to proteins through reactive nitrene intermediates that are generated by low intensity
ultraviolet light r & 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 can be used as
antibody binding agents.
l 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-3a-6a-dipheny1glycouril-3 attached to the
binding agent (e.g., antibody) (US. Pat. Nos. 4,472,509 and 4,938,948, each incorporated
herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the
presence of a coupling agent such as glutaraldehyde or periodate. Protein binding (e.g.,
antibody) conjugates with fluorescein markers are prepared in the presence of these coupling
agents or by on with an isothiocyanate. In US. Pat. No. 948, imaging of breast
tumors, for example, is ed using monoclonal dies, and the detectable imaging
es are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-
succinimidyl(4-hydroxyphenyl)propionate.
In other embodiments, derivatization of immunoglobulins by selectively
introducing sulfhydryl groups in the Fc region of an immunoglobulin using reaction
conditions that do not alter the dy combining site are plated. Antibody
conjugates ed according to this methodology are disclosed to exhibit improved
longevity, specificity and sensitivity (US. Pat. No. 5,196,066, orated 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).
In other embodiments of the invention, immunoglobulins are radiolabeled with
nuclides such as tritium. In additional ments, nanogold particles (such as sizes from
about 0.5 nm-40 nm) and/or Quantum Dots (Hayward, Calif.) are employed.
When a sandwich assay format is used, the capture antibody will be unlabeled.
The detection dy will be either directly labeled, or detected indirectly by addition (after
washing off excess detection antibody) of a molar excess of a second, labeled antibody
directed against the first antibody.
The label used for the detection antibody is any detectable fianctionality that
does not ere with the g of the FOLRl antibodies. Examples of suitable labels are
those numerous labels known for use in immunoassay, including moieties that may be
detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as
moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of
such labels include the radioisotopes 32F, 14C, 125I, 3
H, and 131I, fluorophores such as rare
earth chelates or cein and its derivatives, rhodamine and its derivatives, dansyl,
iferone, luciferases, e.g., firefly luciferase and bacterial luciferase (US. Pat. No.
4,737,456), luciferin, hydrophthalazinediones, horseradish peroxidase (HRP), alkaline
atase, B-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose
oxidase, galactose oxidase, and glucose—6—phosphate dehydrogenase, heterocyclic oxidases
such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen
peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase,
/avidin, biotin/streptavidin, biotin/Streptavidin—B—galactosidase with MUG, spin labels,
bacteriophage labels, stable free radicals, and the like. As noted herein, the fluorimetric
detection is one example.
Conventional methods are ble to bind these labels covalently to proteins
or polypeptides. For instance, coupling agents such as dialdehydes, carbodiimides,
dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the
antibodies with the herein-described fluorescent, chemiluminescent, and enzyme labels. See,
for example, US. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al.
Nature 144:945 ; David et al. Biochemistry 13:1014-1021 (1974); Pain et al. J.
Immunol. Methods 40:219-230 (1981); and Nygren J. Histochem. and Cytochem. 30:407-412
(1982). In certain embodiments, labels herein are fluorescent to increase amplification and
sensitivity to 8 pg/ml, more preferably biotin with streptavidin—B-galactosidase and MUG for
ying the . In certain embodiments, a colorimetric label is used, e.g., where the
detectable antibody is biotinylated and the detection means is avidin or streptavidinperoxidase
and 3,3',5,5'-tetramethyl benzidine.
The conjugation of such label, including the enzymes, to the antibody is a
standard manipulative procedure for one of ry skill in immunoassay techniques. See,
for example, ivan et al. "Methods for the ation of Enzyme-antibody ates
for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. J. Langone and H. Van
Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.
Following the addition of last labeled antibody, the amount of bound antibody
is ined by removing excess unbound labeled dy through washing and then
measuring the amount of the attached label using a detection method appropriate to the label,
and correlating the measured amount with the amount of shed FOLRl in the biological
sample. For example, in the case of enzymes, the amount of color ped and measured
will be a direct measurement of the amount of shed FOLRl present. Specifically, if HRP is
the label, the color can be detected using the substrate 3,3',5,5'—tetramethyl benzidine at 450
nm absorbance.
IX. Substrates and Indicators
The use of substrates and indicators is contemplated for detection of FOLRI.
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 s, HRP and some ke ties 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 ibly
inhibited. It is the excess hydrogen peroxide and the absence of an electron donor that brings
about quenching of endogenous HRP activities. When used in assay systems, HRP can also
be used to convert a defined substrate into its activated chromagen, thus causing a color
change. The HRP enzyme can be conjugated to an antibody, peptide, polymer, or other
molecule by a number of methods that 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, ing in much more efficient labeling 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 substrates, for example. Conjugation with biotin also
involves two steps, as biotin must first be tized to the biotinyl-N-hydroxysuccinimide
ester or to biotin ide before it can be reacted with the epsilonamino groups of the HRP
enzyme.
iaminobenzidine (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 sing its staining intensity and electron density. Of the several metals
and methods used to intensify the l density of polymerized DAB, gold chloride in
combination with silver sulfide appears to be the most successful.
3-Aminoethylcarbazole (AEC), is a substrate for enzymes such as HRP,
and upon oxidation, forms a rose-red end product that is alcohol soluble. Therefore,
specimens processed with ABC must not be immersed in alcohol or alcoholic solutions (e.g.,
Harris' hematoxylin). d, an aqueous rstain and mounting medium should be used.
4-Chloronaphthol (CN) is a substrate for enzymes such as HRP that
precipitates as a blue end product. e CN is soluble in l and other organic
ts, the specimen must not be dehydrated, exposed to alcoholic counterstains, or
coverslipped with mounting media containing c solvents. Unlike DAB, CN tends to
diffuse from the site of precipitation.
p-Phenylenediamine dihydrochloride/pyrocatechol (Hanker-Yates reagent) is a
substrate for enzymes such as HRP that gives a lack on 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 ques.
Calf intestine alkaline phosphatase (AP) (molecular weight 100 kD) removes
(by hydrolysis) and ers phosphate groups from organic esters by breaking the P-O bond;
an intermediate enzyme-substrate bond is briefly formed. The chief metal activators for AP
are Mg++, Mn++ and Ca++.
AP had not been used extensively in immunohistochemistry until publication
of the unlabeled alkaline phosphatase—anti—alkaline 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 ed to the
peroxidase anti-peroxidase (PAP) technique is the lack of interference posed by endogenous
peroxidase activity. Endogenous peroxidase can be blocked using a dilute solution of
hydrogen peroxide. 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 ate on, although 5
mM has been found to be more effective. Intestinal alkaline phosphatases are not adequately
inhibited by levamisole.
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 (chrornogen) to produce ble, colored azo dyes.
Several different combinations of substrates and chromogens have been used successfully.
Naphthol AS-MX phosphate AP substrate can be used in its acid form or as
the sodium salt. The chromogen substrate Fast Red TR and Fast Blue BB produce a bright
red or blue end t, respectively. Both are soluble in alcoholic and other c
solvents, so aqueous mounting media must be used. Fast Red TR is preferred when ng
cell smears.
Additional exemplary substrates include naphthol AS-BI phosphate, naphthol
AS-TR phosphate and 5-bromochloroindoxyl phosphate (BCIP). Other possible
chromogens include Fast Red LB, Fast Garnet GBC, Nitro Blue Tetrazolium (NBT) and
iodonitrotetrazolium Violet (INT), for example.
X. Immunodetection Methods
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. Some
immunodetection s include immunohistochemistry, flow cytometry, enzyme linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay,
fluoroimmunoassay, uminescent 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 0, Methods Mol Biol.
09:215-37; Gulbis B and Galand P, Hum Pathol. 1993 Dec;24(12):l27l-85; and De
Jager R et al., Semin Nucl Med. 1993 Apr;23(2):l65-79, each incorporated herein by
reference.
In general, the immunobinding methods include obtaining a sample ted
of sing ligand protein, polypeptide and/or peptide, and contacting the sample with a
first ligand binding agent (e.g., an anti-ligand dy) in accordance with the t
invention, as the case may be, under conditions effective to allow the formation of
immunocomplexes.
In terms of antigen ion, the biological sample ed may be any
sample in which it is desirable to detect FOLRl such as fluidic extract, blood, plasma, serum,
spinal fluid, lymph fluid, tissue section or specimen, homogenized tissue extract, biopsy
aspirates, a cell, separated and/or purified forms FOLRl-containing compositions, or any
biological fluid. In some embodiments, blood, , or lymph samples or extracts are
used.
Contacting the chosen biological sample with the antibody under ive
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 ns
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.
In l, 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 tic tags. US. s concerning the use of such labels
include US. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 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 ement, as is known in the art.
The anti-ligand antibody employed in the detection may itself be linked to a
detectable label, wherein one would then simply detect this label, thereby allowing the
amount of the primary immune complexes in the composition to be determined.
Alternatively, the first antibody that s bound within the primary immune complexes
may be ed 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. The primary immune xes are contacted with the labeled, secondary binding
agent, or antibody, 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 d secondary antibodies or
ligands, and the remaining label in the secondary immune complexes is then detected.
Further methods e the detection of primary immune complexes by a
two-step approach. A second binding agent, such as an dy, that has binding affinity for
the antibody is used to form secondary immune complexes, as described herein. 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 able label, ng
detection of the tertiary immune complexes thus formed. This system may provide for signal
amplification if this is desired.
In another embodiment, a biotinylated monoclonal or polyclonal dy is
used to detect the target n(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 n is present,
some of the antibody binds to the n to form a biotinylated antibody/antigen complex.
The antibody/antigen complex is then ed by incubation in successive ons of
streptaVidin (or avidin), biotinylated DNA, and/or complementary ylated 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 conjugate can be produced that is copically visible.
In one embodiment, immunohistochemistry (IHC) is used for immunological
detection. Using IHC, detection of FOLRl in a sample can be achieved by ing a sample
with a probe e.g., an anti-FOLRl antibody. The probe can be linked, either directly or
indirectly to a detectable label or can be detected by another probe that is linked, either
directly or indirectly to a detectable label.
In some embodiments, IHC can distinguish n different levels of protein
expression, e.g., calibrated IHC. In some ments, the IHC can distinguish staining
intensity for samples having low FOLRl, intermediate FOLR], or high FOLRl expression.
In one embodiment, immunological detection (by immunohistochemistry) of
FOLRl is scored for both intensity and uniformity (percent of stained cells — membrane
only). Comparative scales for FOLRI expression for intensity correlate as 0 — Negative, 0-1
- Very Weak, l — Weak, 1—2 — Weak to Moderate, 2 — Moderate, 2-3 - te to Strong, 3
— Strong, 3+ - Very Strong. Quantitatively, Score 0 represents that no membrane staining is
observed. Score 1 represents that a faint/barely perceptible membrane staining is ed.
For Score 2, a weak to moderate complete membrane staining is observed. Lastly, Score 3
(or 3+) represents that moderate to complete ne staining is observed. Those samples
with 0 or 1 score for FOLRl expression can be characterized as not having ed FOLRl
expression, whereas those samples with 2 or 3 scores can be characterized as overexpressing
or having elevated FOLRl. In another embodiment, using the antibodies, antigen-binding
fragments thereof, or polypeptides provided herein, those samples with a 0 score for FOLRl
expression can be characterized as not having ed FOLRl expression, those samples
with a 1 score can be characterized as having increased expression of FOLRl, and those
samples with 2 or 3 scores can be terized as overexpressing or having elevated FOLRl.
Samples overexpressing FOLR] can also be rated by immunohistochemical
scores corresponding to the number of copies of FOLR] molecules expressed per cell, or
antibodies bound per cell (ABC), and can been ined biochemically. Comparative
scales for FOLRl uniformity (percent cell membrane staining) are as follows: Negative =
0%; Focal = <25%; heterogeneous (hetero) = 25—75%, and homogeneous (homo) = >75%.
In one embodiment, immunological detection (by immunohistochemistry) of
FOLRl is scored using H-scores. H-scores combine staining ity scores (e. g., a score of
0 to 3, wherein 0 represents no staining, and 3 represents strong staining) with the percentage
of cells that are positive for membrane staining (i.e., uniformity). An H-score can be
cacluated as follows:
H score = [0*(percentage of cells staining at ity 0)] + [1 *(percentage of cells staining at
ity 1)] + rcentage of cells staining at intensity 2)] + [3*(percentage of cells
ng at intensity 3)]. Accordingly, an H-score can range from 0 (no cell membranes
staining) to 300 (all cell membranse staining at intensity 3).
In one embodiment, a t having cancer is identified as a candidate for
treatment with an anti-FOLRl treatment regimen (e.g., IMGN853) when the H-score for
FOLR expression in a tumor sample from the subject is at least 50. In one embodiment, a
subject having cancer is identified as a ate for treatment with an anti-FOLRI treatment
regimen (e.g., IMGN853) when the H-score for FOLR expression in a tumor sample from the
subject is at least 75. In one embodiment, a subject having cancer is identified as a candidate
for treatment with an anti-FOLRl treatment regimen (e.g., IMGN853) when the H-score for
FOLR expression in a tumor sample from the subject is at least 100. In one embodiment, a
subject having cancer is identified as a candidate for treatment with an anti-FOLRl treatment
regimen (e.g., IMGN853) when the H—score for FOLR sion in a tumor sample from the
subject is at least 125. In one embodiment, a subject having cancer is fied as a
candidate for ent with an anti-FOLRl treatment regimen (e. g., IMGN853) when the H-
score for FOLR sion in a tumor sample from the subject is at least 150. In one
embodiment, a subject having cancer is identified as a candidate for treatment with an anti-
FOLRl treatment regimen (e.g., IMGN853) when the H-score for FOLR expression in a
tumor sample from the subject is at least 175. In one embodiment, a subject having cancer is
identified as a candidate for treatment with an OLRI treatment regimen (e.g.,
IMGN853) when the H—score for FOLR expression in a tumor sample from the subject is at
least 200. In another ment, a subject having cancer is identified as a candidate for
treatment with an anti-FOLRl regimen (e.g., IMGN853) when the H-score for FOLR
expression in a tumor sample from the subject is at least 225. In another ment, a
subject having cancer is fied as a candidate for treatment with an OLRl regimen
(e.g., IMGN853) when the H—score for FOLR sion in a tumor sample from the subject
is at least 250. In another embodiment, a subject having cancer is fied as a candidate
for treatment with an anti-FOLRI regimen (e.g., IMGN853) when the e for FOLR
expression in a tumor sample from the t is at least 275. In another embodiment, a
subject having cancer is identified as a candidate for treatment with an anti-FOLRl regimen
(e.g., 3) when the H-score for FOLR expression in a tumor sample from the subject
is 300.
In another embodiment, a subject having ovarian cancer is identified as a
candidate for treatment with an anti-FOLR] regimen (e.g., IMGN85 3) when the H—score for
FOLR expression in an ovarian tumor sample from the subject is 75 to 300. In another
embodiment, a subject having ovarian cancer is identified as a candidate for treatment with
an anti-FOLRl regimen (e.g., IMGN853) when the H-score for FOLR expression in an
ovarian tumor sample from the subject is at least 75. In r embodiment, a subject
having ovarian cancer is identified as a candidate for treatment with an anti-FOLRI regimen
(e.g., IMGN853) when the H-score for FOLR expression in an ovarian tumor sample from
the subject is at least 100. In another ment, a subject having ovarian cancer is
identified as a candidate for treatment with an anti-FOLR] regimen (e.g., IMGN853) when
the H-score for FOLR expression in an ovarian tumor sample from the subject is at least 125.
In another embodiment, a subject having ovarian cancer is identified as a candidate for
treatment with an anti—FOLRI regimen (e.g., 3) when the H—score for FOLR
expression in an ovarian tumor sample from the subject is at least 150. In r
embodiment, a subject having ovarian cancer is identified as a candidate for treatment with
an anti-FOLRl regimen (e.g., IMGN853) when the H—score for FOLR expression in an
ovarian tumor sample from the subject is at least 175. In another embodiment, a subject
having ovarian cancer is identified as a candidate for ent with an anti-FOLRl regimen
(e.g., IMGN853) when the H-score for FOLR expression in an ovarian tumor sample from
the subject is at least 200. In another embodiment, a subject having ovarian cancer is
identified as a candidate for treatment with an anti-FOLRl regimen (e.g., IMGN853) when
the H-score for FOLR expression in an ovarian tumor sample from the subject is at least 225.
In r embodiment, a subject having ovarian cancer is identified as a candidate for
ent with an anti-FOLRl regimen (e.g., IMGN853) when the e for FOLR
expression in an ovarian tumor sample from the subject is at least 250. In another
embodiment, a subject having ovarian cancer is identified as a candidate for treatment with
an anti-FOLRl regimen (e.g., IMGN853) when the H-score for FOLR sion in an
ovarian tumor sample from the subject is at least 275. In another embodiment, a subject
having ovarian cancer is identified as a candidate for treatment with an anti-FOLRl regimen
(e.g., IMGN853) when the H-score for FOLR expression in an ovarian tumor sample from
the subject is 300.
In another ment, a subject having NSCLC is identified as a candidate
for treatment with an anti-FOLRl regimen (e.g., IMGN853) when the H—score for FOLR
expression in an NSCLC tumor sample from the subject is 50 to 300. In another
embodiment, a subject having NSCLC is identified as a candidate for treatment with an anti-
FOLRl regimen (e.g., IMGN853) when the H-score for FOLR expression in an NSCLC
tumor sample from the subject is at least 50. In another embodiment, a subject having
NSCLC is identified as a candidate for treatment with an anti-FOLRl regimen (e.g.,
3) when the H-score for FOLR expression in an NSCLC tumor sample from the
subject is at least 75. In another ment, a subject having NSCLC is identified as a
candidate for treatment with an anti-FOLR] n (e.g., IMGN85 3) when the H-score for
FOLR expression in an NSCLC tumor sample from the subject is at least 100. In another
embodiment, a subject having NSCLC is identified as a candidate for treatment with an anti-
FOLRl regimen (e.g., IMGN853) when the e for FOLR expression in an NSCLC
tumor sample from the t is at least 125. In another ment, a subject having
NSCLC is identified as a candidate for treatment with an OLRl regimen (e.g.,
IMGN853) when the H—score for FOLR expression in an NSCLC tumor sample from the
subject is at least 150. In another embodiment, a subject having NSCLC is identified as a
candidate for treatment with an anti—FOLRl regimen (e.g., IMGN85 3) when the H-score for
FOLR expression in an NSCLC tumor sample from the subject is at least 175. In r
embodiment, a subject having NSCLC is identified as a candidate for treatment with an anti-
FOLRl regimen (e.g., IMGN853) when the H-score for FOLR sion in an NSCLC
tumor sample from the subject is at least 200. In r embodiment, a subject having
NSCLC is identified as a candidate for treatment with an anti-FOLRl regimen (e.g.,
3) when the H-score for FOLR expression in an NSCLC tumor sample from the
subject is at least 225. In another embodiment, a subject having NSCLC is identified as a
candidate for treatment with an anti-FOLR] regimen (e.g., IMGN85 3) when the H-score for
FOLR expression in an NSCLC tumor sample from the subject is at least 250. In another
ment, a subject having NSCLC is identified as a candidate for treatment with an anti-
FOLRI n (e.g., IMGN853) when the H-score for FOLR expression in an NSCLC
tumor sample from the subject is at least 275. In another ment, a subject having
NSCLC is identified as a candidate for treatment with an anti-FOLRl regimen (e.g.,
3) when the H-score for FOLR expression in an NSCLC tumor sample from the
t is 300.
In another embodiment, a subject having endometrial cancer is identified as a
candidate for treatment with an anti-FOLRl regimen (e.g., IMGN85 3) when the H—score for
FOLR expression in an endometrial tumor sample from the subject is 50 to 300. In another
embodiment, a subject having endometrial cancer is identified as a candidate for treatment
with an anti-FOLRl regimen (e.g., IMGN853) when the H-score for FOLR expression in an
endometrial tumor sample from the subject is at least 50. In another embodiment, a t
having endometrial cancer is identified as a candidate for treatment with an anti-FOLRl
regimen (e.g., IMGN853) when the H-score for FOLR expression in an endometrial tumor
sample from the subject is at least 75. In r embodiment, a subject having endometrial
cancer is identified as a candidate for treatment with an anti-FOLRl regimen (e.g.,
IMGN853) when the H-score for FOLR expression in an endometrial tumor sample from the
t is at least 100. In another embodiment, a subject having endometrial cancer is
identified as a candidate for treatment with an anti-FOLR] regimen (e.g., IMGN853) when
the e for FOLR expression in an endometrial tumor sample from the subject is at least
125. In another embodiment, a subject having endometrial cancer is identified as a candidate
for treatment with an anti-FOLRI regimen (e.g., IMGN853) when the H-score for FOLR
expression in an trial tumor sample from the subject is at least 150. In another
embodiment, a subject having endometrial cancer is identified as a candidate for treatment
with an anti-FOLRl regimen (e.g., IMGN853) when the H-score for FOLR expression in an
trial tumor sample from the subject is at least 175. In another embodiment, a subject
having endometrial cancer is identified as a candidate for treatment with an anti-FOLRl
regimen (e.g., IMGN853) when the H-score for FOLR expression in an endometrial tumor
sample from the subject is at least 200. In another embodiment, a subject having endometrial
cancer is identified as a candidate for ent with an anti-FOLRl regimen (e.g.,
IMGN853) when the e for FOLR sion in an endometrial tumor sample from the
subject is at least 225. In another embodiment, a subject having endometrial cancer is
identified as a candidate for ent with an anti-FOLR] regimen (e.g., IMGN853) when
the H-score for FOLR expression in an endometrial tumor sample from the subject is at least
250. In another ment, a subject having endometrial cancer is identified as a candidate
for treatment with an anti-FOLRI regimen (e.g., IMGN853) when the H-score for FOLR
expression in an endometrial tumor sample from the subject is at least 275. In another
embodiment, a t having endometrial cancer is identified as a candidate for treatment
with an anti-FOLRl regimen (e.g., IMGN853) when the H-score for FOLR expression in an
endometrial tumor sample from the subject is 300.
By way of example, an e in a subject having ovarian cancer may be as
follows:
H score = (75% at intensity 0) + (0% at intensity 1) + (0% at intensity 2) + (25% at intensity
3) = 75; or
H score = (0% at intensity 0) + (75% at intensity 1) + (0% at intensity 2) + (25% at intensity
3) = 150.
In another example, an H-score in a subject having endometrial cancer may be as follows:
H score = (75% at intensity 0) + (0% at intensity 1) + (25% at intensity 2) + (0% at intensity
3) = 50; or
H score = (0% at intensity 0) + (75% at intensity 1) + (25% at intensity 2) + (0% at intensity
3) = 125.
In all four examples above, the subject could be identified as a candidate for treatment with
an anti-FOLRl treatment regimen (e.g., 3).
In one embodiment, immunological detection (by histochemistry) of
FOLRl is scored using percent positivity and intensity across a . In this embodiment,
ion for treatment with an anti—FOLRl treatment regimen is based on the percentage of
cells in a sample that are found to express membrane FOLRl at a specified level that reflects
both the staining ity (e.g., 1, 2, or 3) and uniformity (e.g., heterogeneous or
homogeneous (see Table 11)). For example, a sample having at least 25% (i.e., 25-75% or >
75%) of the cells staining for FOLRl positivity at 3 could be characterized as "3 hetero" and
"3 homo" or, collectively, as "at least 25% positive at 3."
In one embodiment, a t having ovarian cancer is identified as a
ate for treatment with an anti—FOLR] treatment regimen (e.g., IMGN853) when at
least 25% of the FOLRl membrane expression in a tumor sample from the subject has an
intensity score of 3 by IHC. In one embodiment, the IHC is med using the FOLR1-2.1
antibody.
In r embodiment, a t having endometrial cancer is identified as a
candidate for treatment with an OLR] treatment regimen (e.g., IMGN853) when at
least 25% of the FOLR membrane expression in a tumor sample from the subject has an
intensity score of at least 2 by IHC. In one embodiment, the IHC is performed using the
FOLR1-2.1 antibody.
In another embodiment, a subject having NSCLC is identified as a candidate
for ent with an anti-FOLRl treatment regimen (e.g., IMGN853) when at least 25% of
the FOLR membrane expression in a tumor sample from the subject has an intensity score of
at least 2 by IHC. In one example, the IHC is peformed using the FOLR1-2.1 antibody for
IHC can be med manually or using an automated system (e.g., using an
automated stainer). IHC can be performed on cells, cell pellets, tissues, preparations from
blood, plasma, serum, or lymph fluid, etc. In some embodiments, the samples are fixed
samples. In some embodiments, the samples are paraffin embedded samples. In some
embodiments, the samples are formalin fixed and paraffin ed samples.
In one embodiment, flow cytometry is used for immunological detection.
Thus, for e, the number of antibodies bound per cell (ABC) can be assessed using
flow cytometry. A high number of anti-FOLRl antibodies bound per cell can indicate high
FOLRl expression levels and a high likelihood to be susceptible to ent with an anti-
FOLRl antibody or immunoconjugate thereof.
XI. Compositions and Kits
Also provided by the invention are compositions and 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 dy (as well as
the marker itself), s, and/or reagents and instrumentation for the isolation nally 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 ion 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.
In still r embodiments, the present invention concerns immunodetection
kits for use with the immunodetection s described . As the antibodies are
generally used to detect FOLRI, he antibodies will generally be included in the kit. The
immunodetection kits will thus comprise, in suitable container means, a first antibody that
binds to FOLRl and/or optionally, an immunodetection reagent and/or further optionally, a
FOLRl protein or cell sample containing FOLRl.
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 dies
that have binding affinity for the first antibody.
Further suitable immunodetection reagents for use in the present kits include
the two-component reagent that comprises a secondary dy that has binding affinity for
the first antibody, along with a third antibody that has binding affinity for the second
antibody, the third antibody being linked to a detectable label. As noted herein, 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.
The kits may further comprise a therapeutic agent for the treatment of cancer,
such as an anti-FOLRl immunoconjugate.
The kit may further comprise an a FOLR] ion reagent used to measure
FOLRI sion in a subject sing a FOLRI detection reagent, and instructions for
use. In one embodiment, the FOLRI detection reagent comprises a FOLR1 binding peptide
or anti- FOLRl antibody. In another embodiment, the kit further ses a secondary
antibody which binds the anti— FOLRl antibody.
In one embodiment the FOLRl—specific antibody is included at a
concentration of about 0.1 to about 20 ug/mL, about 0.1 to about 15 ug/mL, about 0.1 to
about 10 ug/mL, about 0.5 to about 20 ug/mL, about 0.5 to about 15 ug/mL, about 0.5 to
about 10 ug/mL, about 1 to about 20 ug/mL, about 1 to about 15 ug/mL, about 1 to about 10
ug/mL, about 2 to about 20 ug/mL, about 2 to about 15 ug/mL, or about 2 to about 10
ug/mL. In another embodiment, the specific antibody is included at a concentration
of about 1.5 ug/mL, about 2 ug/mL, about 3 ug/mL, about 4 ug/mL, about 5 ug/mL, about 6
ug/mL, about 7 ug/mL, about 8 ug/mL, about 9 ug/mL, or about 10 ug/mL. In another
embodiment, the FOLRl-specific dy is included at a concentration of about 2 ug/mL.
In another embodiment, the FOLRl—specific dy is included at a concentration of about
ug/mL.
In another embodiment, the antibody is included in concentrated on with
instructions for dilutions to achieve a final concentration of about 1 to about 20 ug/mL, about
1 to about 15 ug/mL, about 1 to about 10 ug/mL, about 2 to about 20 ug/mL, about 2 to
about 15 ug/mL, or about 2 to about 10 ug/mL. In another ment, the antibody is
included in concentrated solution with instructions for dilutions to achieve a final
concentration of about 1.5 ug/mL, about 2 ug/mL, about 3 ug/mL, about 4 ug/mL, about 5
ug/mL, about 6 ug/mL, about 7 ug/mL, about 8 ug/mL, about 9 ug/mL, or about 10 ug/mL.
In another embodiment, the antibody is included in trated solution with instructions
for dilutions to achieve a final concentration of about 2 ug/mL. In another embodiment, the
antibody is included in concentrated solution with instructions for dilutions to achieve a final
concentration of about 10 ug/ml.
In another embodiment, the kit further comprises a detection reagent selected
from the group consisting of: an enzyme, a fluorophore, a radioactive label, and a
phore. In another embodiment, the detection reagent is selected from the group
consisting of: biotin, digoxigenin, fluorescein, tritium, and rhodamine.
The kit can also include ctions for ion and scoring of FOLRl
expression. The kit can also include control or reference samples. Non-limiting examples of
control or reference samples include cell s or tissue culture cell lines derived from
normal (normal l) or tumor (positive control) samples. ary cell lines include
cell lines stably or transiently transfected with an expression vector that expresses FOLRl.
Additional examples include cell pellets and tissue samples described in the Examples.
In some embodiments, a kit is a packaged combination including the basic
ts of: (a) capture ts comprised of the monoclonal antibodies against human
FOLRl; and (b) detection reagents which can also comprise FOLRl monoclonal antibodies,
but can also comprise detectable (labeled or unlabeled) antibodies that bind to FOLRl. These
basic elements are defined herein.
In one embodiment, the kit further comprises a solid t for the capture
reagents, which can be provided as a separate element or on which the capture reagents are
already immobilized. Hence, the capture antibodies in the kit can be immobilized on a solid
t, or they can be immobilized on such t that is included with the kit or provided
separately from the kit.
In one embodiment, the capture reagent is coated on a iter plate. The
detection reagent can be labeled antibodies detected directly or unlabeled antibodies that are
detected by labeled antibodies ed against the unlabeled dies raised in a different
species. Where the label is an enzyme, the kit will ordinarily include substrates and ors
required by the enzyme, and where the label is a fluorophore, a dye sor that provides
the detectable chromophore. Where the detection reagent is led, the kit can further
comprise a detection means for the detectable antibodies, such as the labeled antibodies
directed to the unlabeled antibodies, e.g., in a fluorimetric-detected format. Where the label is
an enzyme, the kit will ordinarily include substrates and cofactors required by the enzyme,
where the label is a fluorophore, a dye precursor that provides the detectable chromophore,
and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated
to HRP or B-galactosidase with MUG.
In one embodiment, the e reagent is the FOLRl antibody 2.1, 5.7, or
9.20 or an antibody comprising the sequences of antibody 2.1, 5.7 or 9.20. In one
embodiment, the detection reagent is the FOLRl antibody 2.1, 5.7, or 9.20 or an dy
comprising the sequences of antibody 2.], 5.7 or 9.20. In another embodiment, the detection
reagent FOLRl antibody 2.1, 5.7, or 9.20 or an dy comprising the sequences of
antibody 2.1, 5.7 or 9.20 is biotinylated.
The kit also typically contains instructions for carrying out the assay, and/or
FOLRl protein, or fragments thereof (e.g., FOLRl extracellular domain or the FOLRl
extracellular domain and all or a part of the GP] linkage domain) as an antigen standard, as
well as other additives such as stabilizers, washing and incubation buffers, and the like. In
one embodiment, the FOLRl antigen standard is a FOLRl-Fc immunoadhesin. The kit can
also e ctions for detection and scoring of FOLR] expression.
The components of the kit can be provided in predetermined ratios, with the
ve amounts of the various reagents ly varied to provide for concentrations in
solution of the reagents that substantially maximize the sensitivity of the assay. Particularly,
the reagents can be provided as dry powders, usually lyophilized, including excipients, which
on dissolution will provide for a reagent solution having the appropriate concentration for
combining with the sample to be tested.
Compositions comprising the antibodies or antigen-binding fragments
described herein are also ed. In one embodiment, a composition comprises an anti-
FOLRl antibody or antigen-binding fragment bed herein and a , e.g., a buffer that
can be used in a detection assay such as FACS, IHC, or ELISA. Such buffers are known to
those of ordinary skill in the art and include diluents. By way of example, certain FACS
buffers are provided , e.g., in the working examples. FACS buffers can also contain,
for example, serum or albumin (such as calf serum, goat serum, or BSA) and/or sodium
azide. FACS buffers can also contain PBS, EDTA, and/or DNAse or any combination
thereof. IHC buffers are also provided herein and known to those of ordinary skill in the art.
IHC buffers can contain, for example, casein serum or albumin (such as calf serum, goat
serum, or BSA), Tween or Triton, PBS and/or sodium azide or any combination f.
ELISA buffers are also provided herein and known to those of ordinary skill in the art.
ELISA buffers can contain, for e, serum or albumin (such as calf serum, goat serum,
or BSA), non-fat dry milk, casein, and/or gelatin or any combination thereof.
Embodiments of the present disclosure can be further defined by reference to
the following miting es, which be 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 s, can be practiced without departing from the scope of the present
disclosure.
EXAMPLES
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
ted to persons skilled in the art and are to be included within the spirit and purview of
this application.
Example 1: Generation of FOLRl hybridomas
Hybridomas producing anti—human FOLRl onal antibodies that are
suitable for immunohistochemistry (IHC) staining (antibodies of the invention) were selected
from more than 16,000 hybridomas. The hybridomas were produced by immunization of
wild-type Balb/c mice with different antigens including: formalin fixed 300-19 cells that have
been transfected with human FOLRl, human FOLRl-murine IgGZa Fc recombinant protein,
and human FOLRl recombinant protein. The immunization with fixed 300-19 cells was
ted by subcutaneous injection of transfected 300-19 cells in PBS (5E6
cells/mouse/injection) in the absence of any adjuvant. The immunization with FOLR1
recombinant proteins was done by subcutaneous ion of the protein emulsified in
complete Freund’s adjuvant (CPA) or incomplete Freund’s adjuvant for boost (Sigma) or
Magic mouse adjuvant (Creative Diagnostics). Generally, mice were immunized five times
with two week intervals before receiving a final boost by intraperitoneal injection of the
immunogen three days prior to fusion.
A total of 16 independent s (including fusions 352, 353, and 354) were
carried out using spleen cells that originated from the immunized ype Balb/c mice and
murine myeloma P3X63Ag8.653 cells (P3 . Cell fusion was conducted using an
ECM200 electrofusion machine (BTX Harvard Apparatus) according to standard ols.
Each fusion yielded more than 1,000 hybridomas. Antibodies produced by these hybridomas
were screened and confirmed by a FACS based method using denatured FOLRl-positive and
FOLRl-negative cells. Of the greater than 16,000 hybridomas screened, 14 hybridomas that
were positive by FACS screening were discovered. All of the positive hybridomas originated
from mice immunized with human FOLRl—murine IgG2a Fc recombinant protein.
Of the 14 hybridomas which were initially positive by FACS ing, only
ten showed a sufficient IgG tration for further analysis.
Example 2. Immunohistochemical evaluation of hybridoma supematants
Ten of the initial 14 hybridomas were analyzed by IHC. The analysis was
performed using the Leica Bond RX Automated Stainer and the reagents and conditions
listed in Table 9.
Table 9. IHC Reagents and Assay Conditions
Action/Reaent (Vendor)
ture: 60°C
Dewax Bond Dewax on (Leica) Fixed
100% l Pharmco Aaer
Antigen Retrieval Bond e Retrieval 2 20 Minutes
(ethylenediaminetetraacetic acid
based oH 9.0 solution
nous Peroxidase Peroxide (Leica) 5 Minutes
Block
Test Article ImmunoGen, Inc. generated 15 Minutes
antibodies at varying
concentrations prepared by
diluting in Leica Antibody
Detection
Hematox lin Leica
Slides containing formalin fixed paraffin embeded (FFPE) cells, normal
tissues, patient lung tumor biopsies, and t ovarian tumor biopsies were baked at 60°C
and dewaxed using Bond Dewax Solution and 100% Ethanol. Heat induced epitope retrieval
using Bond e Retrieval 2 (ethylenediaminetetraacetic acid based pH 9.0 solution) was
performed for 20 minutes and endogenous peroxidase was blocked with peroxide for 5
minutes. Slides were incubated with ImmunoGen, Inc. generated antibodies or
Leica/Novocastra mngGl control antibodies at varying concentrations for 15 minutes.
Bound antibodies were detected by incubation with the Leica Bond Refine detection system.
Following the application of the antibodies, slides were incubated with Post Primary Reagent
(rabbit anti-mouse IgG) for 8 minutes, Polymer (goat anti-rabbit polymer) for 8 minutes, and
DAB (3,3-diaminobenzidine tetrahydrochloride) for 10 minutes which resulted in a brown
color signal. Slides were counterstained with hematoxylin for 5 s.
FFPE tissue samples were derived from human tissue blocks obtained from
Proteogenex and the Cooperative Human Tissue Network (CHTN) as outlined below. FFPE
cell s were derived from the KB cell line supplied by American Tissue Culture
tion. Slides containing sections of samples were ed from FFPE blocks using a
microtome set at Sum and were mounted on positively charged slides. These slides were
d to air dry overnight prior to staining.
Table 10. FFPE Test Samples
Normal ry Gland CHTN
Ovarian Papillary Serous Adenocarcinoma
Lung Adenocarcinoma CHTN
FOLRl staining intensity and distribution patterns were scored relative to
control IgG staining (non-specific). Intensity was scored on a scale of 0 to 3 where 0 = no
staining, 1 = weak staining, 2 = te staining, and 3 = strong staining. Uniformity of the
staining was scored as negative (no cells exhibit positive staining), focal (<25% of cells
stained), heterogeneous (25-75% of cells d), and homogeneous (>75% of cells stained).
The staining ity and scoring scales are bed below. All staining was evaluated by
a Board certified pathologist.
Table 11. Intensity and Uniformity of Staining
Intensity (Amount of Membrane
Uniformity (Percent of Positive Cells)
Heterogeneous (hetero) 25-75%
IHC Selection Process to Identify Hybridoma(s) for FFPE FOLR1 IHC
Primary clones positive by FACS on FOLRl-positive denatured cells (ten
clones total) were evaluated by IHC. Two clones were ed from fusion 352 (clones
352.1 and 352.2). Six clones were obtained from fusion 353 (clones 353.1, 353.2, 353.3,
353.5, 353.9, 353.15), and two clones were obtained from fusion 354 (clones 354.1 and
354.2). Hybridoma tants were collected from the cultured hybridoma cells and used
for the analysis. Antibody concentrations in hybridoma supematants were determined by
ELISA using urine L-chain specific polyclonal antibody to capture murine monoclonal
antibody from supernatant and anti-murine Fc—specific polyclonal antibody to detect the
captured antibody; murine monoclonal IgG] sample with know tration was used as a
standard to calculate IgG concentration. Cell culture media (undiluted) was shown not to
interfere with IHC staining s (no background/non-specific staining was noted when
media was used in place of the primary antibody). Ten supematants (IMGN 352.1, 352.2,
353.1, 353.2, 353.3, 353.5, 353.9, 353.15, 354.1, and 354.2) diluted at varying concentrations
up to 10 ug/mL in Leica Antibody Diluent were stained using FOLRl known positive control
samples (human normal lung, patient derived ovarian serous papillary adenocarcinoma, and
KB cells) and evaluated to identify positive candidate clones (clones depicting acceptable
membrane staining and specificity in FOLRl positive samples). Five of the ten clones
exhibited able membrane staining in FOLRl positive samples and good specificity. A
suitable staining concentration was experimentally determined for each of the five candidate
clones as follows: 353.1 (0.7 ug/mL), 353.2 (2.3 ug/mL), 353.3 (2.3 , 353.5 (2 ug/mL
and 10 ug/mL), and 353.9 (2 ug/mL and 10 ug/mL). Of the remaining 5 clones, clone 353.15
(stained at 2 and 10 ug/mL) exhibited acceptable membrane staining in KB cells and normal
lung tissue; however, cytoplasmic staining only was observed in the patient ovarian tumor
tissue tested. Clones 352.1, 352.2, and 354.1 exhibited no visible staining in any samples and
clone 354.2 exhibited only nt non-specific cytoplasmic staining, all considered
unacceptable.
The five candidate clones were r subcloned. Subclones for four clones
(353.2, 353.3, 353.5 and 353.9) were successfully identifiedno nes of clone 353.1 was
generated. A total of eight subclones were purified. Two subclones were obtained from clone
353.5 (353.5-7 and 353.5-10). Two subclones were obtained from clone 353.9 (353.9-20 and
353.9-21). Two subclones were obtained from clone 353.3 (353.3-8 and 353.3-9), and two
subclones were obtained from clone 353.2 (353.2—1 and 353.2-12). (Note that these subclones
are also referred to as 5.7, 5.10, 9.20, 9.21, 3.8, 3.9, 2.1, and 2.12, respectively.) IHC
characterization of the subclones was performed using methods described above (Table 9:
IHC Reagents and Assay Conditions) at antibody concentrations of 2 and 10 ug/mL. The
eight subclones were also sequenced as bed in Example 3, below. The ate
subclones were fiarther evaluated to identify and rank for optimal membrane staining and
specificity as follows: [353.2-1, 353.2-12], [353.9-20, 353.9-21], [353.5-7,353.5-10], and
[353.3-8, 353.3-9] and were selected for r characterization (subclones are bracketed
together according to ce identity as described in e 3).
Two subclones were selected for further IHC assay optimization: 353.2-1 and
. Both antibodies were used to stain human normal lung, human normal salivary
gland, human normal pancreas, patient ovarian cancer biopsies, t non-small cell lung
cancer (NSCLC) biopsies, and patient clear cell renal cell oma biopsies. At optimal
conditions (see Table 12 below and Figure 13), both subclones exhibited specific and
appropriately sensitive ng in both human normal and patient tumor tissues. Ducts of
pancreas, respiratory epithelium of normal lung, and alated ducts exhibited positive
membrane associated staining. Acinar cells/islets of pancreas, interalveolar connective tissue
of lung, and acinar cells of salivary gland expected to be negative did not exhibit positive
ng with either subclone. Tumor cells from ovarian cancer, NSCLC, and clear cell renal
—lOO—
cell carcinoma samples expected to be positive exhibited positive membrane associated
staining that was localized to the tumor cells. Tumor substructures ed to be negative
(stroma, vessels, and lymphocytes) did not exhibit positive staining with 353.2-1 or 353.9-20.
Additional staining of normal tissues with 353-2.] -2. l) are summarized in Table 13,
below and shown in Figure 14. Taken er, the IHC characterization data suggests that
3532-1 and 3539-20 are specific to FOLRl in FFPE tissues (see Figure l and Figure 2).
Table 12. Optimized Assay Conditions
Action/Rea-ent Vendor
Temerature: 60°C
Dewax Bond Dewax Solution (Leica) Fixed
100% Ethanol (Pharmco Aaper)
n Retrieval Bond Epitope Retrieval 2 20 Minutes
(ethylenediaminetetraacetic acid
oH 9.0 solution
Block
IMGN353.9—20 at 6.0 ; mL
Detection
Mixed DAB Leica 10 Minutes
Counterstain 5 s
Table 13. Optimized Assay Conditions
Normal Tissue, Structure 2.1 Staining
Adrenal Gland
Breast lobules
Fallopian tube, surface epithelium
Kidney, tubules
Pancreas, ducts
Pituitary, pituitary cells
Liver, hepatocytes —
—lOl—
Pancreas, acinar cells
Stomach, surface lium, pits
Example 3. Characterization of the selected anti—FOLR1 antibodies
As described above in Example 2, of the fourteen oma clones selected
based on primary and confirmation FACS screening, ten primary clones were analyzed by
immunohistochemistry (IHC) analysis. Of the ten primary clones (i.e., 352.1, 352.2, 353.1,
353.2, 353.3, 353.5, 353.9, 353.15, 354.1, and 354.2), five were positive by IHC (i.e., 353.1,
353.2, and 353.3, 353.5, and 353.9), and all five were derived from the same fusion (fusion
353). Four of the five were successfully ned. One subclone of primary clone 353.2
was chosen and named 353.2—l ("2.1"). One subclone of primary clone 353.3 was chosen
and named 353.3-8 ("3.8"). One subclone of primary clone 353.5 was chosen and named
353.5-7 ("5.7"), and two subclones of primary clone 353.9 were chosen and named 353.9-20
("9.20") and 353.9-21 "). Subclones 9.20 and 9.21 were sequenced, and as expected,
both subclones had the same sequence. In addition, two of the clones, 2.1 and 9.20 were
deposited with ATCC as PTA-120197 and PTA—120196, respectively, on April 16, 2013.
Specificity of the anti-FOLR1 antibodies by Western blot
Specificity of the generated antibodies was analyzed by Western blot with a
panel of cell lysates prepared from FOLR1—positive (Igrov-l, Ovcar-3, Caov-3, Wish, and
Skov-3) and FOLR1-negative (BxPC3, Panc-l, and ASPCl) cell lines. For the assay, lysates
were run in SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose
ne by the rd ures. The membrane was incubated with the anti-FOLR1
antibodies of the ion, and the formed antigen-antibody complexes were detected with
secondary anti-murine antibodies conjugated with horse-radish peroxidase (hrp) (Figure 3).
A11 tested anti-FOLR1 dies ized FOLR1 in cell lines with high levels of FOLR1
expression (i.e., Igrov-l and Wish). FOLR1 in low expressing cell lines Ovcar—3, Caov-3 and
Skov-3 was detected only by OLR1 clones 2.1 and 9.21; clones 3.8 and 5.7 did not
stain these cell lysates perhaps due to cient sensitivity of the antibodies. No additional
non-specific bands were detected in FOLR1-positive cell lines by the clones; no staining of
FOLR1-negative cell lines was observed.
—102—
Binding of the anti-FOLRl dies to denatured and not denatured cells
The ability of the anti—FOLRl antibodies to bind to denatured and nondenatured
(native confirmation) FOLRl was assayed by indirect FACS with positive
cells KB and T47D. Cells were harvested by Versine and washed with phosphate buffered
saline (PBS). Denatured cells were prepared by incubation of the cells in PBS containing
% formaldehyde at 4 OC overnight followed by washing with PBS and tion at 95 °C
for 30 min. Denatured and non-denatured cells were then incubated with anti-FOLRl
antibodies diluted in FACS buffer (RPMI-1640 medium supplemented with 2% normal goat
serum) on ice for 2 hours. The cells were centrifuged, washed with PBS and ted for 40
min with FITC-conjugated goat anti-mouse IgG-antibody. The cells were fuged again,
washed with PBS and resuspended with 0.2 ml of PBS containing 1% formaldehyde. Cell-
associated fluorescence was measured using a FACSCalibur flow cytometer with the HTS
multiwell and analyzed using CellQuest Pro (BD Biosciences, San Diego, US). As shown on
Figure 4, all anti-FOLRl antibodies bound to both denatured and not denatured cells.
y ofthe anti-FOLRl dies by ELISA
The binding affinity of the anti-FOLRl antibodies was examined by ELISA
where recombinant humanFOLRl-murine Fc2a n was used as the antigen. The
recombinant protein was immobilized on microtiter plates, and the antibodies were added at a
range of concentrations to the . The plates were incubated for two hours at room
temperature, washed with PBS supplemented with 0.05% Tween-20, and incubated with hrp-
labeled goat anti-murine secondary antibody for one hour at room temperature. The plates
were washed with een-20 again, and bound hrp—conjugated antibody was detected by
adding the hrp-substrate TMB (Bio-EX). Representative results are shown in Figure 5. The
anti-FOLRl antibodies had similar affinity to human FOLRl at half-maximal effective
concentration (EC50) of 0.5 to 0.9 nM.
No cross-reactivity of the anti-FOLRI antibodies with FOLR2 and FOLR3
FOLRl is a member of Folate Receptor family. Cross-reactivity of the anti-
FOLRl antibodies with the other members of the family FOLR2 and FOLR3 was assayed by
ELISA. Recombinant protein His or FOLR3—His (R&D Systems) was immobilized
to Ni-NTA plates (QIAGEN) and the anti—FOLRl antibodies were added to the plates and
incubated for 2 hours at room ature. As positive controls for FOLR2 and FOLR3
ELISA polyclonal anti-FOLR2 and FOLR3 antibodies (R&D systems), respectively, were
—103—
used. The formed antibody-antigen complexes were detected with hrp-labeled goat anti-
murine secondary antibody. As shown in Figure 6, the anti-FOLRl antibodies of the
invention did not bind to FOLR2 or FOLR3; only the control antibodies detected
corresponding antigens.
Example 4. Antigen epitope characterization
Human FOLR1 has three potential sites for N-glycosylation at positions 69,
161 and 201 (UniProt), and, as reported in literature, all three sites are glycosylated. To
characterize the nature of the epitopes ized by the anti-FOLRl antibodies described
herein, g experiments were performed with deglycosylated and non-treated receptor.
Of the generated anti—FOLRl clones, only clone 2.1 was used in the study because, based on
the sequencing data, the clones are related and likely to bind to the same epitope. In addition
to clone 2.1, two other anti-FOLRl antibodies were included: huMovl9 (W0 201 28)
and clone BN3.2 (Leica). In order to deglycosylate FOLRl, recombinant human FOLRl or
lysates of FOLRl-positive KB or Igrov-l cells were treated with a e of osylation
enzymes (Enzymatic DeGlycoMX Kit, QA—bio) ing to the Manufacturer’s protocol.
Then, samples of treated and non-treated FOLRl were used in ELISA and n blot
analysis. For the ELISA, deglycosylated and non-treated FOLRl were immobilized to ELISA
plates (Immulon), and the anti-FOLRl antibodies FRIHC2-l ("2.1") or huMovl9 were
added. After 2 h incubation, antibody-antigen complexes were detected with hrp-labeled goat
anti-human (for huMovl9) or anti-murine (for 2.1) secondary antibody (Figure 7). For the
Western blot is, samples of deglycosylated and non-treated lysates or huFOLRl
recombinant protein were separated by SDS polyacrylamide gel electrophoresis and
transferred to a nitrocellulose membrane by the standard procedures. The ne was
incubated with the anti-FOLRl antibodies 2.], huMovl9, or BN3.2, and the antigen-antibody
complexes were detected with the appropriate secondary anti-murine or anti human
antibodies conjugated with horse-radish peroxidase (Figure 8). As shown in Figures 7 and 8,
binding of antibody 2.1 to deglycosylated vs. non-treated FOLRl was significantly reduced
suggesting the antibody binds to a glycodependent e. In contrast, the other two anti-
FOLRl antibodies, huMovl9 and BN3.2, bind rly to deglycosylated and non-treated
receptor indicating that (i) the FOLRl protein was not d during the deglycosylation
ure and (ii) 9 and BN3.2 recognize protein epitopes of FOLRl.
—104—
Example 5. Cloning and cing of the VL and VH regions of the anti-human FOLRl
antibodies
Total cellular RNA was prepared from 5 x 106 cells of the FOLRl hybridomas
described in Example 1 using an RNeasy kit (QIAgen) according to the manufacturer’s
protocol. cDNA for the eight subclones clones (2.1, 2.12, 3.8, 3.9, 5.7, 5.10, 9.20, and 9.21)
was uently synthesized from total RNA using the SuperScript 11 cDNA synthesis kit
(Invitrogen).
The PCR procedures for amplifying the antibody variable region cDNAs
derived from hybridoma cells were based on methods described in Wang et a1. ((2000) J
Immunol Methods. 233:167-77) and Co et al. ((1992) J Immunol. 148:1149-54). The
le light chain (VL) and le heavy chain (VH) ces were amplified by
degenerate primers on the S'end and either murine kappa or IgGl constant region specific
primers respectively on the 3' end. The PCR reactions were then run on a 1% low melt
e gel, followed by the excision of the 300 to 400 bp amplicon bands that were
uently purified using Zymo DNA mini columns. The purified amplicons were sent to
Beckman r Genomics for sequencing utilizing the same 5' and 3' primers of the PCR
reactions in order to generate the variable region cDNA sequences from both ions.
Since the degenerate primers used to clone the VL and VH cDNA sequences
alter the 5' end, additional sequencing efforts were needed to verify the te cDNA
sequences. The preliminary sequences were entered into a search query of the NCBI IgBlast
site (www.ncbi.nlm.nih.gov/igblast/) to identify the murine germline sequences from which
the antibody sequences had been derived. PCR primers were then designed to anneal to the
germline linked leader sequence of the murine antibody so that this new PCR reaction would
yield a complete variable region cDNA sequence, unaltered by the PCR primers. The PCR
reactions, band purifications, and sequencing were med as described above.
Mass determination for sequence confirmation
The variable regions cDNA ces obtained for each of the anti-FOLRl
antibodies were combined with germline constant region sequences to obtain full length
antibody cDNA sequences. The lar weights of the heavy and light chains were then
calculated from translations of the cDNA sequences and compared with the molecular
weights obtained by LC/MS analyses of the purified murine anti-FOLRl antibodies. The
LC/MS is done by deglycosylating and reducing the antibody to isolate full chain light and
heavy chain peptides. The observed molecular weights for each of the heavy chains matched
—105—
the expected, but each of the light chains was off by approximately 85 Da. Subsequent
peptide fragmentation analysis by LC/MS of the light chain fragments indicated that the final
serine of light chain leader e was in fact retained on the mature light chain, adding
about 87 Da to the expected MW, thus confirming the cDNA sequences for each of the
FOLRI dies.
Composite CDR sequences for the anti-FOLRI antibodies
Alignments of the antibody sequences for the 8 subclones revealed that 3 of
the 4 original hybridomas had produced closely related, but unique, antibodies. As expected,
each of the 4 sister subclone pairs were cal. In addition two sets of subclones were also
identical ing in the 3 unique antibody sequences (SEQ ID NOsz27-32) (2.1, 5.7, and
9.20). The light and heavy chain variable framework sequences of these 3 unique antibodies
are closely related, but each antibody contains unique CDRs, likely as a result of somatic
amino acid substitutions (see Table 14 below). Because these CDR variants of the murine
anti-FOLRI dies were found to be functionally identical, they e some structural
insight into the sequence flexibility of the CDRS of the anti-FOLRl antibodies of the
invention. Light chain CDRs 2 and 3 were identical in each of the antibodies suggesting that
these tightly conserved CDRS can provide a consistent ural basis for FOLRI binding.
On the other hand, the amino acid tutions in the remaining CDRs, particularly those in
heavy chain CDRs 2 and 3, suggest that these positions are al for refinement of the
affinity and specificity of these antibodies. The specific residue substitutions in these CDR
positions also provide examples of residues that can be incorporated within engineered
versions of these antibodies. Table 14 provides a composite CDR sequence listing compiled
from the anti-FOLRl antibodies of the invention. The composite CDRs fied herein can
be used for the design of recombinant antibodies that would be ed to preserve the
functional attributes of the anti-FOLRI antibodies of the present invention.
Table 14 Composite CDRS
Anti-FOLRI composite CDRS
Light Chain
CDRI: KS[T/S][K/E]SLLNSDGFTYLD (SEQ ID NO:24)
CDR2: LVSNHFS (SEQ ID NO:25)
CDR3: FQSNYLPLT (SEQ ID N0226)
Heavy Chain
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