U.S. patent application number 14/798379 was filed with the patent office on 2016-01-07 for detection and treatment of pancreatic, ovarian and other cd70 positive cancers.
This patent application is currently assigned to SEATTLE GENETICS, INC.. The applicant listed for this patent is SEATTLE GENETICS, INC.. Invention is credited to Maureen Ryan, Maria Leia Smith.
Application Number | 20160003847 14/798379 |
Document ID | / |
Family ID | 41162668 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160003847 |
Kind Code |
A1 |
Ryan; Maureen ; et
al. |
January 7, 2016 |
Detection and Treatment of Pancreatic, Ovarian and Other CD70
Positive Cancers
Abstract
The application provides methods of diagnosis, prognosis,
prophylaxis and treatment of ovarian, pancreatic and other cancers
using antibodies that specifically bind to denatured CD70.
Inventors: |
Ryan; Maureen; (Bellevue,
WA) ; Smith; Maria Leia; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEATTLE GENETICS, INC. |
Bothell |
WA |
US |
|
|
Assignee: |
SEATTLE GENETICS, INC.
Bothell
WA
|
Family ID: |
41162668 |
Appl. No.: |
14/798379 |
Filed: |
July 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12937190 |
Oct 8, 2010 |
9120854 |
|
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PCT/US2009/040275 |
Apr 10, 2009 |
|
|
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14798379 |
|
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61044457 |
Apr 11, 2008 |
|
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Current U.S.
Class: |
424/144.1 ;
435/7.21; 530/387.3; 530/388.22 |
Current CPC
Class: |
G01N 33/566 20130101;
G01N 33/57407 20130101; C07K 2317/56 20130101; C07K 14/70575
20130101; A61P 1/18 20180101; G01N 2333/70575 20130101; G01N 33/53
20130101; G01N 33/6872 20130101; C07K 16/2875 20130101; C07K
2319/01 20130101; A61P 35/00 20180101; C07K 16/18 20130101; G01N
33/574 20130101; A61P 15/00 20180101; C07K 2317/14 20130101; C07K
2317/565 20130101; C07K 2317/24 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 16/28 20060101 C07K016/28 |
Claims
1. A monoclonal antibody that specifically binds to denatured human
CD70 on a sample of formalin fixed, paraffin-embedded cells or
tissues that express human CD70, wherein the monoclonal antibody
comprises three heavy chain complementarity determining regions
(CDRs): heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3,
wherein the heavy chain CDRs are identical to the heavy chain CDRs
of the antibody SG-21.5D12; and three light chain CDRs: light chain
CDR1, light chain CDR2, and light chain CDR3, wherein the light
chain CDRs are identical to the light chain CDRs of the antibody
SG-21.5D12, and wherein the antibody SG-21.5D12 is produced by the
hybridoma deposited with the ATCC and assigned Accession No.
PTA-8734.
2. The monoclonal antibody of claim 1, which is a chimeric or
humanized antibody.
3. The monoclonal antibody of claim 1, that is antibody SG-21.5D12
as produced by the hybridoma deposited with the ATCC and assigned
Accession No. PTA-8734.
4. The monoclonal antibody of claim 1, comprising the heavy chain
variable region of the antibody SG-21.5D12 and the light chain
variable region of the antibody SG-21.5D12.
5. A diagnostic kit, comprising the monoclonal antibody of claim
1.
6. A method of detecting expression of CD70 in a tissue sample of a
patient, comprising obtaining a tissue sample of pancreas, ovary,
lung, larynx, pharynx, breast, or skin from the patient; fixing the
tissue sample and denaturing CD70 in the tissue sample, wherein the
sample is fixed in formalin and embedded in paraffin; contacting
the fixed tissue sample with the monoclonal antibody of claim 1;
and detecting binding of the monoclonal antibody to the fixed
tissue sample to determine whether CD70 is expressed in the
sample.
7. A combination diagnostic and pharmaceutical kit comprising the
monoclonal antibody of claim 1 for use in diagnosis and an antibody
that specifically binds to an extracellular domain of native of
CD70 for use in therapy.
8. The monoclonal antibody of claim 1, wherein the antibody binds
to the extracellular domain of human CD70 protein.
9. The monoclonal antibody of claim 1, wherein the antibody
cross-reacts with a cynomolgous CD70 protein.
Description
CONTINUITY
[0001] This application is a divisional of U.S. application Ser.
No. 12/937,190 filed on Oct. 8, 2010, now allowed, which is a
national stage application under 35 U.S.C. .sctn.371 of PCT Patent
Application No. PCT/US09/40275, filed Apr. 10, 2009, which claims
the benefit of U.S. Provisional Patent Application No. 61/044,457,
filed Apr. 11, 2008, each of these applications is incorporated by
reference herein in its entirety for all purposes.
BACKGROUND
[0002] CD70 is member of the tumor necrosis factor (TNF) family of
cell membrane-bound and secreted molecules that are expressed by a
variety of normal and malignant cell types. CD70 is a transmembrane
type II protein with its carboxyl terminus exposed to the outside
of cells and its amino terminus found in the cytosolic side of the
plasma membrane (Bowman et al., 1994, J. Immunol. 152:1756-61;
Goodwin et al., 1993, Cell 73:447-56). Human CD70 contains a 20
amino acid cytoplasmic domain, an 18 AA transmembrane domain, and a
155 AA extracellular domain with two potential N-linked
glycosylation sites (Bowman et al., supra; Goodwin et al., supra).
Specific immunoprecipitation of radioisotope-labeled
CD70-expressing cells by anti-CD70 antibodies yields polypeptides
of 29 and 50 kDa (Goodwin et al., supra; Hintzen et al., 1994, J.
Immunol. 152:1762-73). Based on its homology to TNF-alpha and
TNF-beta, a trimeric structure is predicted for CD70 (Petsch et
al., 1995, Mol. Immunol. 32:761-72).
[0003] CD70 has limited expression on normal tissues in humans.
This makes CD70 an attractive target for cancer therapies. CD70
expression has been identified, however, on only a small number of
cancers, such as renal cell carcinoma, colon cancer, certain types
of Non-Hodgkin lymphoma and multiple myeloma. CD70 expression on
cancer cells is typically detected using antibodies that bind to
native CD70, such as by immunohistochemistry. Detection of CD70
expression on fixed patient samples has proved problematic, due to
poor quality antibodies that lack sufficient specificity for CD70.
In particular, cross-reactivity and background staining interfere
with detection of CD70 in fixed samples. The present invention
solves this and other needs.
BRIEF SUMMARY
[0004] The invention provides methods of diagnosis, prognosis,
prophylaxis and treatment and monitoring treatment of ovarian,
pancreatic and other cancers using antibodies to CD70. The
invention further provides methods of diagnosis, prognosis,
prophylaxis and treatment and monitoring treatment of lung, head
and neck (larynx or pharynx), melanoma, glioblastoma, multiple
myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, such as follicular
lymphoma, renal cell carcinoma, including clear cell and papillary,
colorectal and bladder carcinomas.
[0005] In one aspect, an antibody is provided that specifically
binds to denatured CD70 relative to binding to native CD70. In some
embodiments, the antibody specifically binds to denatured CD70 on a
fixed, ovarian SK-OV-3 or pancreatic PANC-1 cancer cell line
relative to binding to native CD70. The antibody can be a
monoclonal antibody, such as a chimeric, humanized or human
antibody. Preferably, the antibody binds to denatured CD70 on
formalin-fixed paraffin embedded cells or tissues with a specific
binding that is the same or better than antibody SG-21.1C1 as
produced by the hybridoma deposited with the ATCC and assigned
Accession No. PTA-8733 or antibody SG-21.5D12 as produced by the
hybridoma deposited with the ATCC and assigned Accession No.
PTA-8734. In particular, the non-specific cross-reactivity of the
antibody is less than that of antibody SG-21.1C1 or antibody
SG-21.5D12. In some embodiments, the antibody can compete for
specific binding to denatured CD70 with antibody SG-21.1C1 or with
antibody SG-21.5D12.
[0006] In another aspect, a diagnostic kit is provided that
comprises an antibody that specifically binds to denatured CD70
relative to native CD70. In a related aspect, a method of detecting
expression of CD70 in a tissue sample of a patient is provided. The
tissue sample can be from the pancreas, ovary, lung, larynx,
pharynx, breast, kidney, brain, colon, blood or skin from the
patient. The issue is fixed and the CD70 protein is denatured. The
fixed tissue sample is contacted with an antibody that binds
specifically to denatured CD70 relative to native CD70, and the
binding of the antibody to the fixed tissue sample is detected to
determine whether CD70 is expressed in the sample. Expression of
CD70 on the fixed tissue sample indicates a likelihood the patient
has a CD70 expressing cancer. In some embodiments, the sample is
fixed with formalin and embedded in paraffin.
[0007] In another aspect, a method is provided for diagnosing,
prognosing, determining a treatment protocol or monitoring
treatment of a patient having cancer of the pancreas, ovary, lung,
larynx, pharynx, breast, or skin. The method includes determining
CD70 expression in cells in a sample from the patient's pancreas,
ovaries, lung, larynx, pharynx, breast, or skin of the patient,
wherein the presence of detectable CD70 expression is used in the
diagnosis, prognosis, determining a treatment protocol or
monitoring treatment of the patient. The sample can be a formalin
fixed paraffin embedded sample. The method can further include
administering an effective regimen of a CD70 antibody or CD70
antibody drug conjugate to the patient if the determining step
indicates a detectable level of CD70.
[0008] In another aspect, a method of treating a CD70 positive
cancer is provided. The method includes administering an effective
regimen of a binding agent to CD70 to a patient having cancer of
the pancreas, ovary, lung, larynx, pharynx, breast, or skin having
detectable expression of CD70, wherein the binding agent is an
antibody, antibody derivative or antibody drug conjugate. The
antibody may have effector function. The patient may have
previously undergone treatment by surgery, radiation and/or
chemotherapy with an agent not directed to CD70 without inducing
remission of the cancer. In some embodiments, the antibody is a
chimeric, humanized, or human antibody, such as a chimeric or
humanized form of monoclonal antibody 1F6 or 2F2. The antibody drug
conjugate can include a cytotoxic agent, such as an anti-tubulin
agent, a DNA minor groove binding agent, or a DNA minor groove
alkylating agent. The antibody in the antibody drug conjugate can
be conjugated to the cytotoxic or cytostatic agent via a linker,
such a linker that is cleavable under intracellular conditions.
[0009] In another aspect, a combination diagnostic and
pharmaceutical kit comprising an antibody that specifically binds
to denatured CD70 for use in diagnosis and an antibody that
specifically binds to an extracellular domain of native of CD70 for
use in therapy.
[0010] Aspects of the invention will best be understood by
reference to the following detailed description of the exemplary
embodiments, taken in conjunction with the accompanying drawings,
figures, and tables.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows a Western blot of protein extracts using
antibodies SG-21.1C1 and SG-21.5D12 to detect denatured CD70 in
protein extracts from 786-O, 293F:CD70 transfected cells and 293F
untransfected cells.
[0012] FIG. 2 shows CD70 protein expression on formalin fixed
paraffin embedded (FFPE) pancreatic cell line PANC-1 using the
SG21.1C1 antibody.
[0013] FIG. 3 shows CD70 protein expression in ovarian cell lines
Ovcar-3, SK-OV-3, Ca-Ov-3 and TOV-21G using SG21.1C1 antibody.
[0014] FIG. 4 shows CD70 protein expression in pancreatic tumor
samples as detected by the antibodies SG-21.1C1 or SG-21.5D12
versus control mIgG.
[0015] FIG. 5 shows CD70 protein expression on normal pancreatic
and pancreatic tumor cells as detected by the SG-21.1C1 antibody
using the chromogen Fast Red (darker color).
[0016] FIG. 6 shows an evaluation of CD70 protein expression in
pancreatic cancer cells. The x-axis indicates the CD70 staining
intensity and the y-axis indicates the percentage of the area that
is CD70-positive.
[0017] FIG. 7 shows an evaluation of CD70 protein expression in
ovarian tumors. The x-axis indicates the CD70 staining intensity
and the y-axis indicates the percentage of area that is
CD70-positive.
[0018] FIG. 8 shows an evaluation of the in vitro cytotoxic
activity of various a humanized 1F6 antibody drug conjugates
against an ovarian cancer cell line, SKOV-3.
[0019] FIGS. 9A, 9B and 9C show evaluations of the in vitro
cytotoxic activity of a humanized 1F6 antibody drug conjugate on
the following cell lines: FIG. 9A shows a CD70-transfected
pancreatic cell line, HPAFII; FIG. 9B shows a CD70 transfected
PANC-1 pancreatic cell line; and FIG. 9C shows a CD70 transfected
MiaPaCa-2 cell line.
[0020] FIGS. 10A and 10B show an evaluation of the in vivo efficacy
of a humanized 1F6 antibody drug conjugate on CD70-transfected
MiaPaCa pancreatic carcinoma tumors in nude mice. FIG. 10A shows
median tumor volume plots continued for each group until one or
more animals were euthanized. FIG. 10B shows a Kaplan-Meier curve
showing time for tumor to reach 800 mm.sup.3 for individual animals
in each group.
DEFINITIONS
[0021] Unless stated otherwise, the following terms and phrases as
used herein are intended to have the following meanings.
[0022] The term "antibody" refers to (a) immunoglobulin
polypeptides and immunologically active portions of immunoglobulin
polypeptides, i.e., polypeptides of the immunoglobulin family, or
fragments thereof, that contain an antigen binding site that
immunospecific ally binds to a specific antigen (e.g., CD70), or
(b) conservatively substituted derivatives of such immunoglobulin
polypeptides or fragments that immunospecifically bind to the
antigen (e.g., CD70). Antibodies are generally described in, for
example, Harlow & Lane, Antibodies: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 1988). Unless otherwise apparent
from the context reference to an antibody also includes antibody
derivatives or drug conjugates as described in more detail
below.
[0023] An "antibody derivative" means an antibody, as defined
above, that is modified by covalent attachment of a heterologous
molecule such as, e.g., by attachment of a heterologous
polypeptide, or by glycosylation, deglycosylation, acetylation or
phosphorylation not normally associated with the antibody, and the
like.
[0024] The term "monoclonal antibody" refers to an antibody that is
derived from a single cell clone, including any eukaryotic or
prokaryotic cell clone, or a phage clone, and not the method by
which it is produced. Thus, the term "monoclonal antibody" is not
limited to antibodies produced through hybridoma technology.
[0025] An "antigen" is an entity to which an antibody specifically
binds.
[0026] The term "inhibit" or "inhibition of" means to a reduce by a
measurable amount, or to prevent entirely.
[0027] The term "agent" means an element, compound, or molecular
entity, including, e.g., a pharmaceutical, therapeutic, or
pharmacologic compound. Agents can be natural or synthetic or a
combination thereof. A "therapeutic agent" is an agent that exerts
a therapeutic (e.g., beneficial) effect on cancer cells, either
alone or in combination with another agent (e.g., a prodrug
converting enzyme in combination with a prodrug). Typically,
therapeutic agents useful in accordance with the methods and
compositions described herein are those that exert a cytotoxic or
cytostatic effect.
[0028] "Cytotoxic effect," in reference to the effect of an agent
on a cell, means killing of the cell.
[0029] "Cytostatic effect" means an inhibition of cell
proliferation.
[0030] A "cytotoxic agent" means an agent that has a cytotoxic or
cytostatic effect on a cell, thereby depleting or inhibiting the
growth of, respectively, cells within a cell population.
[0031] The term "deplete," in the context of the effect of a CD70
antibody on CD70-expressing cells, refers to a reduction in the
number of, or elimination of, the CD70-expressing cells.
[0032] The term "functional," in the context of an anti-CD70
antibody or derivative thereof to be used in accordance with the
methods described herein, indicates that the antibody or derivative
thereof is (1) capable of binding to CD70 and/or (2) depletes or
inhibits the proliferation of CD70-expressing cells alone or when
conjugated to a cytotoxic agent.
[0033] The term "prophylaxis" refers to administration of an
anti-CD70 antibody-drug conjugate (ADC) or ADC derivative to a
subject before the onset of a clinical or diagnostic symptom of a
CD70-expressing cancer (e.g., administration to an individual with
a predisposition or at a high risk of acquiring pancreatic or
ovarian cancer) to (a) block the occurrence or onset of the
CD70-expressing cancer, or one or more of clinical or diagnostic
symptoms thereof, (b) inhibit the severity of onset of the
CD70-expressing cancer, or (c) to lessen the likelihood of the
onset of the CD70-expressing cancer.
[0034] The terms "treatment" or "treat" refer to slowing, stopping,
or reversing the progression of a CD70-expressing cancer in a
patient, as evidenced by a decrease or elimination of a clinical or
diagnostic symptom of the disease, by administration of an
anti-CD70 antibody, antibody drug conjugate or ADC derivative to
the subject after the onset of the clinical or diagnostic symptom
of the CD70-expressing cancer at any clinical stage. Treatment can
include, for example, a decrease in the severity of a symptom, the
number of symptoms, or frequency of relapse.
[0035] The term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The term
"pharmaceutically compatible ingredient" refers to a
pharmaceutically acceptable diluent, adjuvant, excipient, or
vehicle with which a CD70 antibody is administered.
[0036] The term "effective amount," in the context of the
administration of a pharmaceutical agent refers to the amount of
the agent that is sufficient to inhibit the occurrence or
ameliorate one or more clinical or diagnostic symptoms of a
CD70-expressing pancreatic or ovarian cancer in a patient. An
effective amount of an agent is administered according to the
methods described herein in an "effective regimen." The term
"effective regimen" refers to a combination of amount of the agent
and dosage frequency adequate to accomplish treatment of a
CD70-expressing cancer.
[0037] The term "patient" includes human and other mammalian
subjects that receive diagnostic, prophylactic or therapeutic
treatment.
[0038] The abbreviation "AFP" refers to
dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-
iamine.
[0039] The abbreviation "MMAE" refers to monomethyl auristatin
E.
[0040] The abbreviation "AEB" refers to an ester produced by
reacting auristatin E with paraacetyl benzoic acid.
[0041] The abbreviation "AEVB" refers to an ester produced by
reacting auristatin E with benzoylvaleric acid.
[0042] The abbreviation "MMAF" refers to
dovaline-valine-dolaisoleunine-dolaproine-phenylalanine.
[0043] The abbreviations "fk" and "phe-lys" refer to the dipeptide
phenylalanine-lysine.
[0044] The abbreviations "vc" and "val-cit" refer to the dipeptide
valine-citrulline.
[0045] Therapeutic agents are typically substantially pure from
undesired contaminants. This means that an agent is typically at
least about 50% w/w (weight/weight) purity, as well as being
substantially free from interfering proteins and contaminants.
Sometimes the agents are at least about 80% w/w and, more
preferably at least 90 or about 95% w/w purity. However, using
conventional protein purification techniques, homogeneous peptides
of at least 99% purity w/w can be obtained.
DETAILED DESCRIPTION
I. General
[0046] The invention provides methods of diagnosis, prognosis,
prophylaxis and treatment and monitoring treatment of ovarian and
pancreatic cancer using antibodies to CD70. The invention further
provides methods of diagnosis, prognosis, prophylaxis and treatment
and monitoring treatment of lung, head and neck (larynx or
pharynx), melanoma, glioblastoma, multiple myeloma, Hodgkin
lymphoma, non-Hodgkin lymphoma, such as follicular lymphoma, renal
cell carcinoma, including clear cell and papillary, colorectal and
bladder carcinomas. The methods are premised in part on the results
presented in the Examples showing that CD70 is expressed at
elevated levels in certain cancers. The elevated expression was
detected in formalin fixed paraffin embedded (FFPE) samples from
ovarian and pancreatic cancer tissues using antibodies that
specifically bind to denatured CD70. Further, elevated expression
of CD70 was also detected in formalin fixed paraffin embedded
(FFPE) samples from other cancer tissues using antibodies against
the denatured extracellular domain of CD70.
[0047] Although practice of the invention is not dependent on
understanding of mechanism, it is believed that the success in
detecting CD70 in FFPE samples of ovarian and pancreatic tissues in
particular, and other cancers in general, resides in the use of
antibodies that preferentially bind to denatured CD70 in such
samples relative to native CD70. Although the frequency of
detectable CD70 in pancreatic and ovarian cancer and/or its level
are not as high as some other cancerous tissues with which CD70 has
been previously associated, they are very specific for cancerous
tissue relative to normal tissue. Thus, in patients having ovarian
cancer or pancreatic cancer in which CD70 is detectable, CD70
presents a particularly useful target for selectively directing
toxicity to cancerous cells. Similarly, in patients having other
cancers (such as lung, head and neck (larynx or pharynx), melanoma,
glioblastoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin
lymphoma, such as follicular lymphoma, renal cell carcinoma,
including clear cell and papillary, colorectal and bladder
carcinomas), the present invention provides a facile way to detect
CD70 expression in fixed samples from such patients.
II. Antibodies to CD70
[0048] The description that follows first considers properties of
antibodies to CD70 applicable to detection of CD70 in ovarian and
pancreatic cancers and treatment thereof, and then focuses on
preferred properties of antibodies for the respective
applications.
[0049] A. Antibodies to CD70 in General
[0050] Anti-CD70 antibodies include monoclonal, chimeric (e.g.,
having a human constant region and mouse variable region),
humanized, veneered, or human antibodies; single chain antibodies,
or the like. The immunoglobulin molecules can be of any type or
class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g.,
IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).
[0051] Anti-CD70 antibodies can be an antigen-binding antibody
fragment such as, a Fab, a F(ab'), a F(ab').sub.2, a Fd chain, a
single-chain Fv (scFv), a single-chain antibody, a disulfide-linked
Fv (sdFv), a fragment comprising either a V.sub.L or V.sub.H
domain, including nanobodies or fragments from camels, llamas or
the like, or fragments produced by a Fab expression library, or a
CD70-binding fragments of any of the above antibodies described
supra. Antigen-binding antibody fragments, including single-chain
antibodies, can comprise the variable region(s) alone or in
combination with the entirety or a portion of the following: hinge
region, CH1, CH2, CH3 and CL domains. Also, antigen-binding
fragments can comprise any combination of variable region(s) with a
hinge region, CH1, CH2, CH3 and CL domains. Typically, the
antibodies are human, rodent (e.g., mouse and rat), donkey, sheep,
rabbit, goat, guinea pig, camelid, horse, or chicken.
[0052] The antibodies can be mono-specific, bi-specific,
tri-specific, or of greater multi-specificity. Multi-specific
antibodies maybe specific for different epitopes of CD70 or may be
specific for both CD70 as well as for a heterologous protein. (See,
e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et
al., 1991, J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893;
4,714,681; 4,925,648; 5,573,920; and 5,601,819; Kostelny et al.,
1992, J. Immunol. 148:1547-1553.) Multi-specific antibodies,
including bi-specific and tri-specific antibodies, useful for
practicing the methods described herein are antibodies that
immunospecifically bind to both CD70 and a second cell surface
receptor or receptor complex, such as an immunoglobulin gene
superfamily member, a TNF receptor superfamily member, an integrin,
a cytokine receptor, a chemokine receptor, a major
histocompatibility protein, a lectin (C-type, S-type, or I-type),
or a complement control protein.
[0053] Anti-CD70 antibodies can also be described in terms of their
binding affinity to CD70, of 10.sup.-7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10
M, 10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, 10.sup.-12 M, 5.times.10.sup.-13 M,
10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 M,
5.times.10.sup.-15 M, or 10.sup.-15 M.
[0054] An anti-CD70 antibody can be a chimeric antibody. A chimeric
antibody is a molecule in which different portions of the antibody
are derived from different animal species, such as antibodies
having a variable region derived from a murine monoclonal antibody
and a human immunoglobulin constant region. Methods for producing
chimeric antibodies are known in the art. (See, e.g., Morrison,
Science, 1985, 229:1202; Oi et al., 1986, BioTechniques 4:214;
Gillies et al., 1989, J. Immunol. Methods 125:191-202; U.S. Pat.
Nos. 5,807,715; 4,816,567; and 4,816,397.)
[0055] An anti-CD70 antibody can also be a humanized antibody
including a veneered antibody. Humanized antibodies are antibody
molecules that bind the desired antigen and have one or more
complementarity determining regions (CDRs) from a non-human
species, and framework and constant regions from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, or preferably
improve, antigen binding. These framework substitutions are
identified by methods well known in the art, e.g., by modeling of
the interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riecbmann et al., 1988, Nature 332:323.) Antibodies can be
humanized using a variety of techniques known in the art such as
CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106;
EP 0 519 596; Padlan, Molecular Immunology, 1991, 28(4/5):489-498;
Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska
et al., 1994, PNAS 91:969-973), and chain shuffling (U.S. Pat. No.
5,565,332) (all of these references are incorporated by reference
herein).
[0056] An anti-CD70 antibody can also be a human antibody. Human
antibodies can be made by a variety of methods known in the art
such as phage display methods (see supra) using antibody libraries
derived from human immunoglobulin sequences. See also, e.g., U.S.
Pat. Nos. 4,444,887 and 4,716,111; WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
In addition, a human antibody recognizing a selected epitope can be
generated using a technique referred to as "guided selection," in
which a selected non-human monoclonal antibody, e.g., a mouse
antibody, is used to guide the selection of a completely human
antibody recognizing the same epitope (see, e.g., Jespers et al.,
1994, Biotechnology 12:899-903). Human antibodies can also be
produced using transgenic mice that express human immunoglobulin
genes. Monoclonal antibodies directed against the antigen can be
obtained from the immunized, transgenic mice using hybridoma
technology. For an overview of the technology for producing human
antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol.
13:65-93. For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;
European Patent No. 0 598, 877; and U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598.
[0057] Antibodies can be assayed for specific binding to CD70 by
known methods, such as for example, competitive and non-competitive
immunoassay systems using techniques such as Western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays. (See, e.g., Ausubel et al., eds., Short Protocols in
Molecular Biology (John Wiley & Sons, Inc., New York, 4th ed.
1999); Harlow & Lane, Using Antibodies: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.,
1999.)
[0058] Further, the binding affinity of an antibody to CD70 and the
off-rate of an antibody CD70 interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
CD70 (e.g., .sup.3H or .sup.125I) with the antibody of interest in
the presence of increasing amounts of unlabeled CD70, and the
detection of the antibody bound to the labeled CD70. The affinity
of the antibody for CD70 and the binding off-rates can then be
determined from the data by Scatchard plot analysis. Competition
with a second antibody can also be determined using
radioimmunoassays. In this case, CD70 is incubated with the
antibody of interest conjugated to a labeled compound (e.g.,
.sup.3H or .sup.125I) in the presence of increasing amounts of an
unlabeled second antibody. Alternatively, the binding affinity of
an antibody to CD70 and the on- and off-rates of an antibody-CD70
interaction can be determined by surface plasmon resonance.
[0059] Antibodies can be made from antigen-containing fragments of
the CD70 protein by standard procedures according to the type of
antibody (see, e.g., Kohler, et al., Nature, 256:495, (1975);
Harlow & Lane, Antibodies, A Laboratory Manual (C.S.H.P., NY,
1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033
(1989) and WO 90/07861; Dower et al., WO 91/17271 and McCafferty et
al., WO 92/01047 (each of which is incorporated by reference for
all purposes). As an example, monoclonal antibodies can be prepared
using a wide variety of techniques including, e.g., the use of
hybridoma, recombinant, and phage display technologies, or a
combination thereof. Hybridoma techniques are generally discussed
in, e.g., Harlow et al., supra, and Hammerling, et al., In
Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier,
N. Y., 1981). Examples of phage display methods that can be used to
make the anti-CD70 antibodies include, e.g., those disclosed in
Briinnan et al., 1995, J. Immunol. Methods 182:41-50; Ames et al.,
1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994,
Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18;
Burton et al., 1994, Advances in Immunology 57:191-280; PCT
Application No. PCT/GB91/01 134; PCT Publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108 (the
disclosures of which are incorporated by reference herein).
[0060] Techniques for generating antibody fragments that recognize
specific epitopes are also generally known in the art. For example,
Fab and F(ab').sub.2 fragments can be produced by proteolytic
cleavage of immunoglobulin molecules, using enzymes such as papain
(to produce Fab fragments) or pepsin (to produce F(ab').sub.2
fragments). F(ab').sub.2 fragments contain the variable region, the
light chain constant region and the C.sub.H1 domain of the heavy
chain. Techniques to recombinantly produce Fab, Fab' and
F(ab').sub.2 fragments can also be employed using, e.g., methods
disclosed in WO 92/22324; Mullinax et al., 1992, BioTechniques
12(6):864-869; and Sawai et al., 1995, AJRI 34:26-34; and Better et
al., 1988, Science 240:1041-1043 (the disclosures of which are
incorporated by reference herein).
[0061] Examples of techniques that can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in
Enzymology 203:46-88; Shu et al., 1993, Proc. Natl. Acad. Sci. USA
90:7995-7999; and Skerra et al., 1988, Science 240:1038-1040.
[0062] Anti-CD70 antibodies and derivatives thereof that are useful
in the present methods can also be produced by recombinant
expression techniques. Recombinant expression of an antibody or
derivative thereof that binds to CD70 and/or depletes or inhibits
the proliferation of CD70-expressing cells requires construction of
an expression vector containing a nucleic acid that encodes the
antibody or derivative thereof. Once a nucleic acid encoding such a
protein has been obtained, the vector for the production of the
protein molecule may be produced by recombinant DNA technology
using techniques well known in the art. Standard techniques such as
those described in Sambrook and Russell, Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 3rd ed., 2001); Sambrook et al., Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 2nd ed., 1989); Ausubel et al., Short Protocols in
Molecular Biology (John Wiley & Sons, New York, 4th ed., 1999);
and Glick & Pasternak, Molecular Biotechnology: Principles and
Applications of Recombinant DNA (ASM Press, Washington, D.C., 2nd
ed., 1998) can be used for recombinant nucleic acid methods,
nucleic acid synthesis, cell culture, transgene incorporation, and
recombinant protein expression.
[0063] For example, for recombinant expression of an anti-CD70
antibody, an expression vector may encode a heavy or light chain
thereof, or a heavy or light chain variable domain, operably linked
to a promoter. An expression vector may include, e.g., the
nucleotide sequence encoding the constant region of the antibody
molecule (see, e.g., WO 86/05807; WO 89/01036; and U.S. Pat. No.
5,122,464), and the variable domain of the antibody may be cloned
into such a vector for expression of the entire heavy or light
chain. The expression vector is transferred to a host cell by known
techniques, and the transfected cells are then cultured to produce
the anti-CD70 antibody. Typically, for the expression of
double-chained antibodies, vectors encoding both the heavy and
light chains can be co-expressed in the host cell for expression of
the entire immunoglobulin molecule.
[0064] A variety of prokaryotic and eukaryotic host-expression
vector systems can be utilized to express an anti-CD70 antibody or
derivative thereof. Typically eukaryotic cells, particularly for
whole recombinant anti-CD70 antibody molecules, are used for the
expression of the recombinant protein. For example, mammalian cells
such as Chinese hamster ovary cells (CHO) (e.g., DG44 or CHO-S) in
conjunction with a vector such as the major intermediate early gene
promoter element from human cytomegalovirus or the Chinese hamster
ovary EF-1.alpha. promoter, is an effective expression system for
the production of anti-CD70 antibodies and derivatives thereof
(see, e.g., Foecking et al., 1986, Gene 45:101; Cockett et al.,
1990, Bio/Technology 8:2; Allison, U.S. Pat. No. 5,888,809).
[0065] Other host-expression systems include, plasmid-based
expression systems in bacterial cells (see, e.g., Ruther et al.,
1983, EMBO 1,2:1791; Inouye & Inouye, 1985, Nucleic Acids Res.
13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
24:5503-5509); insect systems such as the use of Autographa
californica nuclear polyhedrosis virus (AcNPV) expression vector in
Spodoptera frugiperda cells; and viral-based expression systems in
mammalian cells, such as, adenoviral-based systems (see, e.g.,
Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359;
Bittner et al., 1987, Methods in Enzymol. 153:51-544).
[0066] B. Antibodies for Detection of CD70
[0067] Selection of antibodies to CD70 for use in detection methods
depends on whether CD70 is detected by a technique that requires
detection of denatured CD70 or native CD70 (as expressed on cells).
In methods, such as Western blotting or immunohistochemical
detection in which the target CD70 is denatured, it is preferable
to use an antibody that binds to CD70 in denatured form (e.g.,
human or cynomolgus CD70). Typically such antibodies preferentially
bind to the denatured form over the native form (i.e., CD70 as it
occurs in nature or when isolated without exposure to denaturing
conditions, such as solvents, detergents or elevated temperatures
(e.g., over 50.degree. C.)). Such antibodies can be raised using a
denatured CD70 immunogen (e.g., human or cynomolgus CD70), or an
immunogenic fragment thereof from the extracellular portion.
Denaturation can be effected by treating the immunogen with SDS
(e.g., 0.5%) and optionally heating up to 80.degree. C.
Denaturation can also be effected simply by eluting an immunogen
from an SDS gel used to purify the immunogen. Alternatively,
antibodies preferentially binding to denatured CD70 can be produced
using synthetic peptides from the extracellular domain of CD70 as
immunogens. Some such peptides are too short to retain the
conformation of a corresponding segment of the native peptide.
Synthetic peptides can be but do not usually require denaturation
to use as an immunogen.
[0068] Antibodies generated by these or other methods are screened
for preferential binding to denatured CD70 relative to native CD70.
Denatured and native CD70 antigen can be assayed by the same assay
or by different assays. Particularly, if the latter approach is
used, the screening can be performed with a control antibody known
to bind native CD70, such as therapeutic antibodies described below
(e.g., humanized 1F6 or 2F2; see U.S. Patent Application
Publications Nos. 2006-0233794 and 2006-0083736 and International
Patent Publication WO 06/113909). If an antibody shows a higher
ratio of binding to denatured CD70 relative to native CD70 relative
to the ratio of the control antibody, then the antibody
preferentially binds to denatured CD70.
[0069] Preferred antibodies for detection of CD70 in pancreatic and
ovarian cancers are those that specifically bind to CD70 on
pancreatic or ovarian cancer specimens that are fixed with formalin
and embedded in paraffin (FFPE). These antibodies preferentially
recognize epitopes on CD70 that are revealed by the FFPE treatment
relative to native CD70 antigens on untreated pancreatic or ovarian
cancer cells. Such antibodies are referred to as FFPE-specific
anti-CD70 antibodies. Such antibodies lack detectable specific
binding to native CD70. Preferably, the specific binding of the
antibodies is same or better than antibody SG-21.1C1 or SG-21.5D12.
In particular, the antibodies preferably have the same or lower
detectable cross-reactivity to other cellular proteins, as
determined by Western blot or staining of fixed cells, under
specific binding conditions, as compared to antibodies SG-21.1C1 or
SG-21.5D12.
[0070] Preferred antibodies for detection of CD70 in other cancers
(as described infra in the Examples) are those which specifically
bind to CD70 in these cancer specimens that are fixed with formalin
and embedded in paraffin (FFPE). These antibodies preferentially
recognize epitopes on CD70 that are revealed by the FFPE treatment
relative to native CD70 antigens on untreated pancreatic or ovarian
cancer cells. Such antibodies lack detectable specific binding to
native CD70. Preferably, the specific binding of the antibodies is
same or better than antibody SG-21.1C1 or SG-21.5D12. In
particular, the antibodies preferably have the same or lower
detectable cross-reactivity to other cellular proteins, as
determined by Western blot or staining of fixed cells, under
specific binding conditions, as compared to antibodies SG-21.1C1 or
SG-21.5D12.
[0071] Some exemplary FFPE-specific anti-CD70 antibodies are mAbs
SG-21.1C1 (also referred to as SG-21.1C1-B3) and SG-21.5D12.C3
(also referred to as SG-21.5D12.C3). Other preferred antibodies
compete with SG-21.1C1.B3 or SG-21.5D12.C3 for specific binding to
denatured CD70. Other preferred antibodies comprise a heavy chain
comprising the three CDRs from the heavy chain of SG-21.1C1.B3 and
a light chain comprising the three CDRs from the light chain of
SG-21.1C1.B3. Other preferred antibodies comprise a heavy chain
comprising the three CDRs from the heavy chain of SG-21.5D12.C3 and
a light chain comprising the three CDRs from the light chain of
SG-21.5D12.C3. Other preferred antibodies comprise a mature heavy
chain variable region having at least 90% sequence identity to the
mature heavy chain variable region of SG-21.1C1.B3 and a mature
light chain variable region having at least 90% sequence identity
to the mature light chain variable region of SG-21.1C1.B3. Other
preferred antibodies comprise a mature heavy chain variable region
having at least 90% sequence identity to the mature heavy chain
variable region of SG-21.5D12.C3 and a mature light chain variable
region having at least 90% sequence identity to the mature light
chain variable region of SG-21.5D12.C3.
[0072] C. Antibodies to CD70 for Therapeutic Applications
[0073] Antibodies used for therapeutic applications specifically
bind to an extracellular domain of native CD70 antigens on
pancreatic or ovarian cancer cells. Antibodies used for therapeutic
applications can also specifically bind to an extracellular domain
of native CD70 antigen on lung, head and neck (larynx or pharynx),
melanoma, glioblastoma, multiple myeloma, Hodgkin lymphoma,
non-Hodgkin lymphoma, such as follicular lymphoma, renal cell
carcinoma, including clear cell and papillary, colorectal and
bladder carcinomas. The antibodies can be agonistic, non-agonistic
or antagonistic with respective to CD70 binding to its ligand CD27.
Although practice of the invention is not dependent on an
understanding of mechanism, it is believed that the antibodies can
exert a cytotoxic or cytostatic effect either as a result of
binding to CD70 and being internalized within a cell, or by binding
to CD70 and accumulating on the outside of cells. In either event,
the cytotoxic or cytostatic effect can be promoted by conjugating
the antibody to a cytotoxic or cytostatic agent. The cytotoxic or
cytostatic effect exerted from the outside of the cell by an
antibody bound to CD70 can additionally or alternatively be
promoted by an antibody constant (effector) function. The antibody
constant domains mediate various Ig effector functions, such as
participation of the antibody in antibody dependent cellular
cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and/or
antibody dependent cellular phagocytosis (ADCP). Optionally, the
effector function of a CD70-binding agent can be augmented by
several approaches as described in WO2006/113909. The cytotoxic or
cytostatic effect exerted by the antibodies also can be promoted by
blocking interaction of CD70 with its ligand, CD27.
[0074] A preferred anti-CD70 antibody is mAb 1F6 or 2F2, or a
chimeric or humanized forms thereof, as described in WO 2004/073656
and Published US Application No. 2006-0233794 and in WO2006/113909.
A preferred heavy chain mature variable region has the sequence of
SEQ ID NO: 1 and a preferred light chain mature variable region has
the sequence of SEQ ID NO:2.
[0075] Other useful antibodies comprise mature heavy and light
chain variable regions having at least 90% and preferably at least
95% or 99% sequence identity to SEQ ID NO: 1 and 2, respectively.
Guidance as to which residues variable region framework residues
are needed for binding is provided by WO2006/113909. Other useful
anti-CD70 antibodies or derivatives thereof can competitively
inhibit binding of mAb 1F6 or 2F2 to CD70, as determined, for
example, by immunoassay. Competitive inhibition means that an
antibody when present in at least a two fold and preferably
five-fold excess inhibits binding of 1F6 or 2F2 to CD70 by at least
50%, more typically at least 60%, yet more typically at least 70%,
and most typically at least 75%, or the antibody competitively
inhibits binding of 1F6 or 2F2 to CD70 by at least 80%, at least
85%, at least 90%, or at least 95%.
[0076] Other preferred antibodies comprise a heavy chain comprising
the three CDRs from the heavy chain variable region of 1F6 and a
light chain comprising the three CDRs from the light chain variable
of 1F6. Other preferred antibodies comprise a mature heavy chain
variable regions having at least 90% sequence identity to the
mature heavy chain variable region of 2F2 and a mature light chain
variable region having at least 90% sequence identity to the mature
light chain variable region of 2F2. Other preferred antibodies
comprise a mature heavy chain variable regions having at least 90,
95 or 99% sequence identity to the mature heavy chain variable
region of 1F6 and a mature light chain variable region having at
least 90, 95 or 99% sequence identity to the mature light chain
variable region of 1F6. Other preferred antibodies comprise a
mature heavy chain variable region having at least 90, 95 or 99%
sequence identity to the mature heavy chain variable region of 2F2
(SEQ ID NO:3) and a mature light chain variable region having at
least 90, 95 or 99% sequence identity to the mature light chain
variable region of 2F2 (SEQ ID NO:4).
[0077] Numerous other antibodies to CD70 are described in, for
example, U.S. Patent Application Publication No. 2005-0191299; and
International Publication No. WO 07/038637. Other antibodies
binding to an extracellular domain of CD70 can be screened for
suitability either alone or as derivatives and/or conjugates as
described below. Screening can assess internalization into cells
expressing CD70 using labeled antibodies. Screening can also assess
cytotoxicity. Additional screening can be performed on animal
models of pancreatic or ovarian cancer and other cancers. For
example, SKOV-3 ovarian carcinoma cell line, AN3CA endometrial
carcinoma cell line, TOV21G ovarian carcinoma cell, can be used.
Also, PANC 1 and MiaPaca2 pancreatic cell lines can be used.
[0078] A derivative of an anti-CD70 antibody can also be used in
the practice of present methods. Typical modifications include,
e.g., glycosylation, deglycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, and the like. Any of numerous
chemical modifications may be carried out by, for example, specific
chemical cleavage, acetylation, formylation or metabolic synthesis
in the presence of tunicamycin. Additionally, the derivative may
contain one or more non-classical amino acids.
[0079] The antibody derivative can be a multimer, such as a dimer,
comprising one or more monomers, where each monomer includes (i) an
antigen-binding region of an anti-CD70 antibody, or a polypeptide
region derived therefrom (e.g., by conservative substitution of one
or more amino acids), and (ii) a multimerizing (e.g., dimerizing)
polypeptide region, such that the antibody derivative forms
multimers (e.g., homodimers) that specifically bind to CD70.
Typically, an antigen binding region of an anti-CD70 antibody, or a
polypeptide region derived therefrom, is recombinantly or
chemically fused with a heterologous protein, wherein the
heterologous protein comprises a dimerization or multimerization
domain. Prior to administration of the antibody derivative to a
subject for the purpose of treating or preventing CD70-expressing
cancers, the derivative is subjected to conditions that allows
formation of a homodimer or heterodimer. A heterodimer may comprise
identical dimerization domains but different CD70 antigen-binding
regions, identical CD70 antigen-binding regions but different
dimerization domains, or different CD70 antigen-binding regions and
dimerization domains.
[0080] An anti-CD70 antibody derivative can be formed by
conjugating an anti-CD70 antibody to a second antibody (an
"antibody heteroconjugate") (see, e.g., U.S. Pat. No. 4,676,980).
Heteroconjugates useful for practicing the present methods comprise
an antibody that binds to CD70 (e.g., an antibody that has the CDRs
and/or heavy chains of the monoclonal antibodies 1F6 or 2F2) and an
antibody that binds to a surface receptor or receptor complex, such
as an immunoglobulin gene superfamily member, a TNF receptor
superfamily member, an integrin, a cytokine receptor, a chemokine
receptor, a major histocompatibility protein, a lectin (C-type,
S-type, or I-type), or a complement control protein.
[0081] Antibodies to CD70 and their derivatives can be conjugated
to a cytotoxic or cytostatic moiety to form an antibody drug
conjugate (ADC). Particularly suitable moieties for conjugation to
antibodies or antibody derivatives are chemotherapeutic agents,
prodrug converting enzymes, radioactive isotopes or compounds, or
toxins. For example, an anti-CD70 antibody or derivative thereof
can be conjugated to a cytotoxic agent such as a chemotherapeutic
agent, or a toxin (e.g., a cytostatic or cytocidal agent such as,
e.g., abrin, ricin A, pseudomonas exotoxin, or diphtheria
toxin).
[0082] The anti-CD70 antibody or derivative thereof can be
conjugated to a pro-drug converting enzyme. The pro-drug converting
enzyme can be recombinantly fused to the antibody or derivative
thereof or chemically conjugated thereto using known methods.
Exemplary pro-drug converting enzymes are carboxypeptidase G2,
beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase,
.beta.-lactamase, .beta.-glucosidase, nitroreductase and
carboxypeptidase A.
[0083] Techniques for conjugating therapeutic agents to proteins,
and in particular to antibodies, are well-known. (See, e.g., Arnon
et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In
Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy
(Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al.,
"Antibodies For Drug Delivery," in Controlled Drug Delivery
(Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review," in Monoclonal Antibodies '84: Biological And Clinical
Applications (Pinchera et al. eds., 1985); "Analysis, Results, and
Future Prospective of the Therapeutic Use of Radiolabeled Antibody
In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection
And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe
et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT
publication WO 89/12624.)
[0084] The therapeutic agent can be conjugated in a manner that
reduces its activity unless it is cleaved off the antibody (e.g.,
by hydrolysis, by antibody degradation or by a cleaving agent).
Such therapeutic agent is attached to the antibody or derivative
thereof with a cleavable linker that is sensitive to cleavage in
the intracellular environment of the CD70-expressing cancer cell
but is not substantially sensitive to the extracellular
environment, such that the conjugate is cleaved from the antibody
or derivative thereof when it is internalized by the
CD70-expressing cancer cell (e.g., in the endosomal or, for example
by virtue of pH sensitivity or protease sensitivity, in the
lysosomal environment or in the caveolear environment).
[0085] Typically, the ADC comprises a linker region between the
therapeutic agent and the anti-CD70 antibody or derivative thereof.
As noted supra, typically, the linker is cleavable under
intracellular conditions, such that cleavage of the linker releases
the therapeutic agent from the antibody in the intracellular
environment (e.g., within a lysosome or endosome or caveolea). The
linker can be, e.g., a peptidyl linker that is cleaved by an
intracellular peptidase or protease enzyme, including a lysosomal
or endosomal protease. Typically, the peptidyl linker is at least
two amino acids long or at least three amino acids long. Cleaving
agents can include cathepsins B and D and plasmin (see, e.g.,
Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most
typical are peptidyl linkers that are cleavable by enzymes that are
present in CD70-expressing cells. For example, a peptidyl linker
that is cleavable by the thiol-dependent protease cathepsin-B,
which is highly expressed in cancerous tissue, can be used (e.g., a
linker comprising a Phe-Leu or a Gly-Phe-Leu-Gly peptide). Other
such linkers are described, e.g., in U.S. Pat. No. 6,214,345. In
specific embodiments, the peptidyl linker cleavable by an
intracellular protease comprises a Val-Cit linker or a Phe-Lys
dipeptide (see, e.g., U.S. Pat. No. 6,214,345, which describes the
synthesis of doxorubicin with the Val-Cit linker). One advantage of
using intracellular proteolytic release of the therapeutic agent is
that the agent is typically attenuated when conjugated and the
serum stabilities of the conjugates are typically high.
[0086] The cleavable linker can be pH-sensitive, i.e., sensitive to
hydrolysis at certain pH values. Typically, the pH-sensitive linker
is hydrolyzable under acidic conditions. For example, an
acid-labile linker that is hydrolyzable in the lysosome (e.g., a
hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,
orthoester, acetal, ketal, or the like) can be used. (See, e.g.,
U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and
Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989,
Biol. Chem. 264:14653-14661.) Such linkers are relatively stable
under neutral pH conditions, such as those in the blood, but are
unstable at below pH 5.5 or 5.0, the approximate pH of the
lysosome. In certain embodiments, the hydrolyzable linker is a
thioether linker (such as, e.g., a thioether attached to the
therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat.
No. 5,622,929)).
[0087] Other linkers are cleavable under reducing conditions (e.g.,
a disulfide linker). Disulfide linkers include those that can be
formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB
(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-
, SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.
47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody
Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,
Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
[0088] The linker can also be a malonate linker (Johnson et al.,
1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau
et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide
analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).
[0089] The linker also can be a non-cleavable linker, such as an
maleimido-alkylene- or maleimide-aryl linker that is directly
attached to the Drug unit. An active drug-linker is released by
degradation of the antibody.
[0090] Typically, the linker is not substantially sensitive to the
extracellular environment meaning that no more than about 20%,
typically no more than about 15%, more typically no more than about
10%, and even more typically no more than about 5%, no more than
about 3%, or no more than about 1% of the linkers in a sample of
the ADC is cleaved when the ADC or ADC derivative present in an
extracellular environment (e.g., in plasma). Whether a linker is
not substantially sensitive to the extracellular environment can be
determined, for example, by incubating independently with plasma
both (a) the ADC or ADC derivative (the "ADC sample") and (b) an
equal molar amount of unconjugated antibody or therapeutic agent
(the "control sample") for a predetermined time period (e.g., 2, 4,
8, 16, or 24 hours) and then comparing the amount of unconjugated
antibody or therapeutic agent present in the ADC sample with that
present in control sample, as measured, for example, by high
performance liquid chromatography.
[0091] The linker can also promote cellular internalization. The
linker can promote cellular internalization when conjugated to the
therapeutic agent (i.e., in the milieu of the linker-therapeutic
agent moiety of the ADC or ADC derivate as described herein).
Alternatively, the linker can promote cellular internalization when
conjugated to both the therapeutic agent and the anti-CD70 antibody
or derivative thereof (i.e., in the milieu of the ADC or ADC
derivative as described herein).
[0092] A variety of linkers that can be used with the present
compositions are described in WO 2004-010957 have the form
-A.sub.a-W.sub.w--Y.sub.y-- (II)
[0093] wherein:
[0094] -A- is a stretcher unit;
[0095] a is 0 or 1;
[0096] each --W-- is independently an amino acid unit;
[0097] w is independently an integer ranging from 0 to 12;
[0098] --Y-- is a spacer unit; and
[0099] y is 0, 1 or 2.
[0100] Representative linkers are depicted within the square
brackets of Formulas (Ia) and (Ib; see infra), wherein A-, --W--,
--Y--, -D, w and y are as defined above and R.sup.1 is selected
from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8 carbocyclo-,
--O--(C.sub.1-C.sub.8 alkyl)-, -arylene-, --C.sub.1-C.sub.10
alkylene-arylene-, -arylene-C.sub.1-C.sub.10 alkylene-,
--C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8 carbocyclo)-,
--(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10. Ab is antibody.
##STR00001##
[0101] The Amino Acid unit (--W--), if present, links the Stretcher
unit (-A-) to the Spacer unit (--Y--) if the Spacer unit is
present, and links the Stretcher unit to the cytotoxic or
cytostatic agent (Drug unit; D) if the spacer unit is absent.
[0102] If present, --W.sub.w-- is a dipeptide, tripeptide,
tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide,
nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. w is
an integer ranging from 2 to 12.
[0103] The Spacer unit (--Y--), when present, links an Amino Acid
unit to the Drug unit. Spacer units are of two general types:
self-immolative and non self-immolative. A non self-immolative
spacer unit is one in which part or all of the Spacer unit remains
bound to the Drug unit after enzymatic cleavage of an amino acid
unit from the anti-CD70 antibody-linker-drug conjugate or the
drug-linker compound. Examples of a non self-immolative Spacer unit
include a (glycine-glycine) spacer unit and a glycine spacer unit.
When an anti-CD70 antibody-linker-drug conjugate containing a
glycine-glycine spacer unit or a glycine spacer unit undergoes
enzymatic cleavage via a tumor-cell associated-protease, a
cancer-cell-associated protease or a lymphocyte-associated
protease, a glycine-glycine-drug moiety or a glycine-drug moiety is
cleaved from Ab-A.sub.a-W.sub.w--. To liberate the drug, an
independent hydrolysis reaction should take place within the target
cell to cleave the glycine-drug unit bond.
[0104] Alternatively, an anti-CD70 antibody drug conjugate
containing a self-immolative spacer unit can release the drug (D)
without the need for a separate hydrolysis step. In these
embodiments, --Y-- is a p-aminobenzyl alcohol (PAB) unit that is
linked to --W.sub.w-- via the nitrogen atom of the PAB group, and
connected directly to -D via a carbonate, carbamate or ether group.
Other examples of self-immolative spacers include aromatic
compounds that are electronically equivalent to the PAB group such
as 2-aminoimidazol-5-methanol derivatives (see Hay et al., 1999,
Bioorg. Med. Chem. Lett. 9:2237 for examples) and ortho or
para-aminobenzylacetals. Spacers can be used that undergo facile
cyclization upon amide bond hydrolysis, such as substituted and
unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995,
Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1]
and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem.
Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et
al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing
drugs that are substituted at the .alpha.-position of glycine
(Kingsbury, et al., 1984, J. Med. Chem. 27:1447) are also examples
of self-immolative spacer strategies that can be applied to the
anti-CD70 antibody-linker-drug conjugates. Alternatively, the
spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit,
which can be used to incorporate additional drugs.
[0105] Useful classes of cytotoxic agents include, for example,
anthracyclines, antitubulin agents, DNA minor groove binders, DNA
replication inhibitors, chemotherapy sensitizers, or the like.
[0106] Examples of useful classes of cytotoxic agents include
auristatins, camptothecins, duocarmycins, etoposides, maytansinoids
and vinca alkaloids.
[0107] Suitable cytotoxic agents include, for example, auristatins
(e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders
(e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g.,
paclitaxel and docetaxel), vinca alkaloids, doxorubicin,
morpholino-doxorubicin, and cyanomorpholino-doxorubicin.
[0108] The cytotoxic agent can be a chemotherapeutic such as, for
example, doxorubicin, paclitaxel, melphalan, vinca alkaloids,
methotrexate, mitomycin C or etoposide. In addition, potent agents
such as CC-1065 analogues, calicheamicin, maytansine, analogues of
dolastatin 10, rhizoxin, and palytoxin can be linked to the
anti-CD70 antibodies or derivatives thereof.
[0109] In exemplary embodiments, the cytotoxic or cytostatic agent
can be auristatin E or a derivative thereof. Typically, the
auristatin E derivative is, e.g., an ester formed between
auristatin E and a keto acid. For example, auristatin E can be
reacted with paraacetyl benzoic acid or benzoylvaleric acid to
produce AEB and AEVB, respectively. Other typical auristatins
include AFP, MMAF, and MMAE. The synthesis and structure of
auristatin E and its derivatives are described in, for example,
U.S. Patent Application Publication Nos. 2005-0238649 and
2006-0074008.
[0110] The cytotoxic agent can be a DNA minor groove binding agent.
(See, e.g., U.S. Pat. No. 6,130,237.) For example, the minor groove
binding agent can be a CBI compound or an enediyne (e.g.,
calicheamicin).
[0111] The ADC or ADC derivative can comprise an anti-tubulin
agent. Examples of anti-tubulin agents include, but are not limited
to, taxanes (e.g., Taxol.RTM. (paclitaxel), Taxotere.RTM.
(docetaxel)), T67 (Tularik), vinca alkyloids (e.g., vincristine,
vinblastine, vindesine, and vinorelbine), and auristatins (e.g.,
auristatin E, AFP, MMAF, MMAE, AEB, AEVB). (Exemplary auristatins
are shown below in Formulas III-XIII Other suitable antitubulin
agents include, for example, baccatin derivatives, taxane analogs
(e.g., epothilone A and B), nocodazole, colchicine and colcimid,
estramustine, cryptophysins, cemadotin, maytansinoids,
combretastatins, discodermolide, and eleutherobin.
##STR00002## ##STR00003##
[0112] The cytotoxic agent can be a maytansinoid, another group of
anti-tubulin agents. For example, the maytansinoid is maytansine or
a maytansine containing drug linker such as DM-1 or DM-4
(ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.
52:127-131).
[0113] Other binding agents to CD70 can be used as an alternative
to antibodies. Such a CD70-targeting moiety can include one or more
CDRs from an antibody that binds to CD70 and depletes or inhibits
the proliferation of CD70-expressing cells when conjugated to a
cytotoxic agent. Typically, the protein is a multimer, most
typically a dimer.
[0114] Other CD70-targeting moieties can include CD27 and variants
or fragments thereof that bind to CD70. CD70-targeting moieties can
further include peptides, ligands and other molecules that
specifically bind to CD70.
[0115] Other CD70-targeting moieties useful in the methods
described herein can be identified using any method suitable for
screening for protein-protein interactions. Typically, proteins are
initially identified by their ability to specifically bind to CD70,
then their ability to exert a cytostatic or cytotoxic effect on
activated lymphocytes or CD70-expressing cancer cells when
conjugated to a cytotoxic or cytostatic agent. Methods that can be
employed include "interaction cloning" techniques which entail
probing expression libraries with labeled CD70 in a manner similar
to the technique of antibody probing of .lamda.gt11 libraries.
(see, e.g., Blanar and Rutter, 1992, Science 256:1014-1018).
Another method is the two-hybrid system (Chien et al., 1991, Proc.
Natl. Acad. Sci. USA 88:9578-9582) and is commercially available
from Clontech (Palo Alto, Calif.).
[0116] Once a CD70-binding protein is identified, its ability
(alone or when multimerized or fused to a dimerization or
multimerization domain or conjugated to a cytotoxic or cytostatic
moiety) to exert a cytostatic or cytotoxic effect on
CD70-expressing cancer cells (when conjugated to a cytotoxic agent)
is determined in similar fashion to that for an antibody.
III. Detecting CD70
[0117] The samples to be assayed for diagnostic applications can be
obtained by surgical procedures, e.g., biopsy. CD70 is typically
detected by an immuno assay in which a sample containing cells
known or suspected to be from a cancer (e.g., pancreatic or ovarian
cancer) is contacted with an antibody. After contact, the presence
or absence of a binding event of the antibody to the cells in the
specimen is determined. The binding is related to the presence or
absence of the antigen expressed on cancerous cells in this
specimen. Generally, the sample is contacted with a labeled
specific binding partner of the anti-CD70 antibody capable of
producing a detectable signal. Alternatively, the anti-CD70
antibody itself can be labeled. Examples of types of labels include
enzyme labels, radioisotopic labels, nonradioactive labels,
fluorescent labels, toxin labels and chemoluminescent labels.
Detection of a signal from the label indicates the presence of the
antibody specifically bound to CD70 in the sample.
[0118] The sample on which the assay is performed can be fixed or
frozen to permit histological sectioning. Preferably, the excised
tissue samples are fixed in aldehyde fixatives such as
formaldehyde, paraformaldehyde, glutaraldehyde; or heavy metal
fixatives such as mercuric chloride. More preferably, the excised
tissue samples are fixed in formalin and embedded in paraffin wax
prior to incubation with the antibody. An advantage afforded by
formalin-fixed paraffin-embedded (FFPE) specimens is the
preservation of cellular and architectural morphologic detail in
tissue sections (see, e.g., Fox et al., 1985, J. Histochem.
Cytochem. 33:845-853). Optionally, FFPE specimens can be treated
with citrate, EDTA, enzymatic digestion or heat to increase
accessibility of epitopes (see, e.g., Shi et al., 1991, J Histochem
Cytochem. 39:741-748).
[0119] Alternatively, a protein fraction can be isolated from cells
from known or suspected pancreatic or ovarian cancer and analyzed
by ELISA, Western blotting, immunoprecipitation or the like. In
another variation, cells can be analyzed for expression of CD70 by
FACS analysis, preferably in combination with another pancreatic or
ovarian cell marker.
[0120] In a further variation, mRNA can be extracted from cells
from known or suspected to be pancreatic or ovarian cancers. The
mRNA or a nucleic acid derived therefrom, such as a cDNA can then
be analyzed by hybridization to a nucleic probe binding to DNA
encoding CD70.
[0121] In another variation, a pancreatic or ovarian cancer can be
detected in vivo by administering a labeled anti-CD70 antibody to a
patient and detecting the antibody by in vivo imaging.
[0122] Detection of CD70 in tissue samples can be qualitative or
quantitative or both. Qualitative detection means detecting the
presence or absence of CD70 expression. Quantitative expression
means determining a level of expression of expression of CD70. The
presence and/or level of CD70 in a pancreatic or ovarian tissue
sample at issue can (but need not) be determined with respect to
one or more standards. The standards can be historically or
contemporaneously determined. The standard can be, for example, a
pancreatic or ovarian sample known not to be cancerous from a
different subject, a tissue from either the patient or other
subject known not to express CD70, or a pancreatic or ovarian cell
line. The standard can also be the patient sample under analysis
contacted with an irrelevant antibody (e.g., an antibody raised to
a bacterial antigen). Because CD70 is not expressed to any
significant extent in non-cancerous pancreatic or ovarian tissue,
such non-cancerous tissue can be used as a zero (background)
expression standard.
[0123] The presence of detectable signal from binding of an
anti-CD70 antibody to CD70 relative to a standard (if used)
indicates the presence of CD70 in the tissue sample, and the level
of detectable binding provides an indication of the level of
expression of CD70. In assays performed on tissue sections, the
level of expression can be expressed as a percentage of the surface
area of the sample showing detectable expression of CD70.
Alternatively, or additionally, the level (intensity) of expression
can be used as a measure of the total expression in the sample or
of the cells expressing CD70 in the sample.
IV. Diagnosis, Prognosis, Designing and Monitoring Treatment
[0124] Detection of expression of CD70 in a sample of pancreatic or
ovarian tissue is an indication that the sample is cancerous.
Similarly, detection of expression of CD70 in a other patient
sample is an indication that the patient has lung, head and neck
(larynx or pharynx), melanoma, glioblastoma, multiple myeloma,
Hodgkin lymphoma, non-Hodgkin lymphoma, such as follicular
lymphoma, renal cell carcinoma, including clear cell and papillary,
colorectal or bladder carcinoma. Antibodies used for therapeutic
applications specifically bind to an extracellular domain of native
CD70 antigens
[0125] The indication of cancer provided by presence and/or level
of CD70 can be combined with means of diagnosis, such as internal
or external examination of a patient by a physician, X-ray, CT Scan
(Computed Tomography), PET Scan (Positron Emission Tomography),
PET/CT Scan, ultrasound, MRI (Magnetic Resonance Imaging),
endoscopy, ERCP (Endoscopic Retrograde Cholangiopancreatography),
histological examination and tissue culturing in arriving at an
overall diagnosis.
[0126] Perhaps of greatest relevance to the physician, the presence
and level of CD70 provides useful information for designing a
treatment protocol for the patient, and in particular administering
an antibody against CD70, a derivative, an ADC or other binding
agent to a patient. Because of the essential absence of detectable
CD70 expression in normal pancreatic or ovarian tissue, the
presence of this receptor in a cancer provides a target for
therapeutic treatment. The higher the level of CD70 expression
and/or the higher percentage of a tumor expressing CD70, the more
effective treatment is likely to be. Continued analysis of CD70
after treatment provides a means of monitoring whether the
treatment is effective, a reduction in the level of CD70-positive
signal (i.e., as a proxy for the presence of CD70-positive cancer
cells) that the treatment is effective.
V. Patients Amenable to Treatment
[0127] Patients amenable to treatment by the methods usually have
detectable levels of CD70 in their pancreatic, ovarian or other
tissue accompanied by other signs or symptoms of cancer as
described above. A variety of subtypes and stages of both
pancreatic and ovarian cancer exist as described in more detail
below. Sometimes, patients treated by the present methods have
undergone other types of treatment previously (e.g., surgery,
chemotherapy and/or radiation) without inducing remission or even
slowing down the growth of the cancer. In some such patients, the
cancer is refractory to treatment by one of more such
therapies.
[0128] Some patients at risk of pancreatic cancer can also be
treated prophylactically before signs and symptoms of the disease
appear. Such individuals include those having relatives who have
experienced these diseases, and those whose risk is determined by
analysis of genetic or biochemical markers.
[0129] A. Pancreatic Cancer Patients
[0130] Pancreatic cancer is a malignant tumor within the pancreatic
gland. Almost 90% of pancreatic cancer patients are older than 55.
The average age at the time this cancer is found is 72. Risk
factors for pancreatic cancer include: age, male gender, African
ethnicity, smoking, diets high in meat, obesity, diabetes, chronic
pancreatitis (has been linked, but is not known to be causal),
occupational exposure to certain pesticides, dyes, and chemicals
related to gasoline, family history, Helicobacter pylori infection,
gingivitis or periodontal disease. (Pancreatic Cancer. Von Hoff et
al., ed., Maine; 2005.) Only 10 to 15% of pancreatic cancer is
considered hereditary. Some genetic markers that are connected to
pancreatic cancer can include mutations in the PNCA1, PALLD or
BRCA2 gene (see, e.g., Banke et al., 2000, Med. Clin. North Am. 84:
677-690; Meckler et al., 2001, Am. J. Surg. Path. 25: 1047-1053;
Pogue-Geile et al., 2006, PLoS Med. 3: e516; Murphy et al., 2002,
Cancer Res. 62: 3789-3793).
[0131] However, not all patients in the currently recognized risk
categories will develop pancreatic cancers. Many pancreatic cancers
arise "sporadically" (i.e., in patients without family
histories).
[0132] Individuals suffering from pancreatic cancer can be
recognized according to the histology of the tumor, obtained in a
pathology report. Histology dictates many aspects of clinical
treatment, management, and prognosis. There are two main types of
pancreatic cancer based on whether the tumor starts from the
exocrine or endocrine gland of the pancreas. Tumors formed from the
exocrine gland of the pancreas are much more common. About 95
percent of pancreatic tumors are adenocarcinomas. The remaining 5
percent include other tumors of the exocrine pancreas (e.g. serous
cystadenomas), acinar cell cancers, and pancreatic neuroendocrine
tumors (such as insulinomas).
[0133] Endocrine tumors are also called islet cell tumors and are
divided into several sub-types. Most are benign, but there are a
few that are cancerous. A special type of cancer (ampullary cancer)
can occur where the bile duct from the liver and the pancreatic
duct empty into the small intestine. Because this type of cancer
often causes signs such as yellowing of the skin and eyes, it is
usually found at an earlier stage than most pancreatic cancers. The
chances of successful treatment are better for patients suffering
from ampullary cancer.
[0134] Pancreatic cancer staging can be performed according to the
American Joint Committee on Cancer (AJCC) criteria. The cancer
stages are labeled using Roman numerals I through IV, with stage IV
indicating that the cancer has spread and is more serious.
Specifically, stage I pancreatic cancer includes tumors which have
not spread into certain proscribed sensitive areas and which have
no involved regional nodes or distal metastasis. Stage II includes
tumors which have spread into the duodenum, bile duct, or
"peripancreatic" tissues and which have no involved regional nodes
or distal metastasis. Stage III cancer includes tumors which may
have or may not have spread into these areas and which have
involved regional nodes, but which show no evidence of distal
metastasis. Stage IVA includes tumors which have spread into the
stomach, spleen, large bowel or the adjacent large vessels and
which have involved regional nodes, but show no evidence of distal
metastasis. Stage IVB includes pancreatic tumors of any kind with
node status of any kind and with evidence of distal metastasis.
Though referred to, this pancreatic cancer staging system is rarely
used in its pure form because the stages do not fully match patient
prognosis or treatment options. An alternative is the three stage
classification (potentially resectable, locally advanced and
metastatic), which is based on radiological findings. Other
prognosis factors are also considered. The grade of the cancer
which indicates how abnormal the cells look under the microscope is
sometimes listed on a scale from G1 to G4, with G1 cancers looking
the most like normal cells and having the best outlook. For
patients who have surgery, the extent of the resection, i.e.,
whether or not all of the tumor is removed, is also important with
regard to outlook. This is sometimes listed on a scale from R0 to
R2 with R0 indicating that all of tumor that can be seen has been
removed and R2 indicating that some tumor that can be seen can not
be removed.
[0135] Early pancreatic cancer symptoms are non-specific and
varied. Common symptoms include pain in the upper abdomen that
typically radiates to the back and is relieved by leaning forward
(seen in carcinoma of the body or tail of the pancreas), loss of
appetite, significant weight loss and painless jaundice related to
bile duct obstruction (carcinoma of the head of the pancreas).
However, all of these symptoms can have multiple other causes and
are not limited to pancreatic cancer.
[0136] B. Ovarian Cancer Patients
[0137] Ovarian cancer is cancer that begins in the ovaries. Ovarian
cancer usually happens in women over age 50, but it can also affect
younger women. Its cause is unknown. Certain populations such as
Ashkenazi Jewish women are at a higher risk, often at an earlier
age than the general population. Patients with a personal history
of breast cancer or a family history of ovarian, breast, or other
related cancers, especially if at a young age, may have an elevated
risk. A strong family history of uterine cancer, colon cancer, or
other gastrointestinal cancers may indicate the presence of a
syndrome known as hereditary nonpolyposis colorectal cancer (HNPCC,
also known as Lynch II syndrome), which confers a higher risk for
developing ovarian cancer. Cytogenetic studies and loss of
heterozygosity investigations suggest that some genes or
chromosomal regions are involved in ovarian cancer initiation and
progression (see, e.g., Pejovic et al., 1992, Genes Chromosomes
Cancer, 4:58-68; Testa et al., 1994, Cancer Res., 54:2778-2784:
Yang-Feng et al., 1993, Int. J. Cancer, 54:546-551). Genetic
markers of risk toward ovarian cancer include, but are not limited
to mutations in the BRCA1 or the BRCA2 gene (Futreal et al., 1994,
Science, 266:120-122). Patients with strong genetic risk for
ovarian cancer may consider the use of preventative oophorectomy
after completion of child-bearing. Not all women in currently
recognized risk categories will develop ovarian cancers. The
majority of ovarian cancers arise sporadically.
[0138] Individuals suffering from ovarian cancer can be recognized
according to the histology of the tumor, obtained in a pathology
report. Histology dictates many aspects of clinical treatment,
management, and prognosis. There are three main types of ovarian
cancer based on the kind of cells the tumor started from and
whether the tumor is benign or cancerous. Germ cell tumors start
from the cells that produce the eggs. Stromal tumors start from
connective tissue cells that hold the ovary together and produce
the female hormones estrogen and progesterone. Epithelial tumors
start from the cells that cover the outer surface of the ovary.
Most ovarian cancers are epithelial tumors, with a minority of
tumors arising from the germ or stromal cells.
[0139] Ovarian cancer often is primary, but can also be secondary,
the result of metastasis from a primary cancer elsewhere in the
body. For example, from breast cancer, or from gastrointestinal
cancer (in which case the ovarian cancer is a Krukenberg cancer).
Surface epithelial-stromal tumor can originate in the lining of the
abdominal cavity, in which case the ovarian cancer is secondary to
primary peritoneal cancer, but treatment is basically the same as
for primary ovarian cancer of this type.
[0140] In ovarian cancers, the cancer stages are as follows: stage
I is limited to one or both ovaries; stage II involves pelvic
extension or implants; stage III involves microscopic peritoneal
metastases beyond the pelvis; or limited to the pelvis with
extension to the small bowel or omentum; and stage IV involves
distant metastases such as in the liver, or outside the peritoneal
cavity.
[0141] Early ovarian cancer is frequently asymptomatic, or produces
only mild symptoms which might be ignored by the patient because
the symptoms are either vague or non-specific. Symptoms can include
bloating, pelvic or abdominal pain, trouble eating or feeling full
quickly, urinary symptoms, such as urgent or frequent feelings of
needing to go. (See, e.g., Smith et al., 2005, Cancer
104(7):1398-1407; The consensus statement released by the American
Cancer Society, the Gynecologic Cancer Foundation, and the Society
of Gynecologic Oncologists on Jun. 12, 2007.) More than 60% of
patients presenting with this cancer already have stage III or
stage IV cancer, when it has already spread beyond the ovaries.
[0142] C. Other Cancer Patients
[0143] Other patients amenable to treatment by the methods usually
have detectable levels of CD70 in samples of lung, head and neck
(larynx or pharynx), melanoma, glioblastoma, multiple myeloma,
Hodgkin lymphoma, non-Hodgkin lymphoma, such as follicular
lymphoma, renal cell carcinoma, including clear cell and papillary,
colorectal or bladder carcinoma. Such patients may be accompanied
by other signs or symptoms of cancer. Sometimes, patients treated
by the present methods have undergone other types of treatment
previously (e.g., surgery, chemotherapy and/or radiation) without
inducing remission or even slowing down the growth of the cancer.
In some such patients, the cancer is refractory to treatment by one
of more such therapies.
[0144] Some patients at risk of cancer can also be treated
prophylactically before signs and symptoms of the disease appear.
Such individuals include those having relatives who have
experienced these diseases, and those whose risk is determined by
analysis of genetic or biochemical markers.
VI. Methods of Treatment
[0145] The present invention provides methods of treating or
prophylaxis of pancreatic or ovarian cancer by the antibodies,
derivatives and ADC, and other anti-CD70 binding agents
(collectively agents) disclosed herein. The compositions can be
administered to a patient
[0146] Various delivery systems can be used to administer the
agents including intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The agents can be administered, for example by infusion or bolus
injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, and the
like) and can be administered together with other biologically
active agents such as chemotherapeutic agents. Administration can
be systemic or local.
[0147] The agents can be administered by injection, by means of a
catheter, by means of a suppository, or by means of an implant, the
implant being of a porous, non-porous, or gelatinous material,
including a membrane, such as a sialastic membrane, or a fiber.
[0148] Alternatively, the agents can be delivered in a controlled
release system. For example, a pump can be used (see Langer, 1990,
Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng.
14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989,
N. Engl. J. Med. 321:574). Alternatively, polymeric materials can
be used (see Medical Applications of Controlled Release (Langer
& Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled
Drug Bioavailability, Drug Product Design and Performance (Smolen
& Ball eds., Wiley, New York, 1984); Ranger & Peppas, 1983,
Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 71:105.) Other controlled
release systems are discussed, for example, in Langer, supra.
[0149] The agents can be administered as pharmaceutical
compositions comprising a therapeutically or prophylactically
effective amount of the agent and one or more pharmaceutically
compatible ingredients. For example, the pharmaceutical composition
typically includes one or more pharmaceutical carriers (e.g.,
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like). Water is a
more typical carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include, for example, starch, glucose, lactose, sucrose,
gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate, talc, sodium chloride, dried skim milk,
glycerol, propylene, glycol, water, ethanol, and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, pH buffering agents (e.g., amino acids)
and/or solubilizing or stabilizing agents (e.g., nonionic
surfactants such as tween or sugars such as sucrose, trehalose or
the like). These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the nucleic acid or protein, typically in purified form,
together with a suitable amount of carrier so as to provide the
form for proper administration to the patient. The formulations
correspond to the mode of administration.
[0150] Typically, compositions for intravenous administration are
solutions in sterile isotonic aqueous buffer. When necessary, the
pharmaceutical can also include a solubilizing agent and a local
anesthetic such as lignocaine to ease pain at the site of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or a concentrate in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
active agent. When the pharmaceutical is to be administered by
infusion, it can be dispensed with an infusion bottle containing
sterile pharmaceutical grade water or saline. When the
pharmaceutical is administered by injection, an ampoule of sterile
water for injection or saline can be provided so that the
ingredients can be mixed prior to administration.
[0151] The amount of the agent that is effective in the treatment
or prophylaxis of pancreatic or ovarian cancer can be determined by
standard clinical techniques. In addition, in vitro assays may
optionally be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation also depends on the
route of administration, and the stage of pancreatic or ovarian
cancer, and should be decided according to the judgment of the
practitioner and each patient's circumstances. Effective doses may
be extrapolated from dose-response curves derived from in vitro or
animal model test systems. A dose can be formulated in animal
models to achieve a circulating plasma concentration range that
includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture.
[0152] For example, toxicity and therapeutic efficacy of the agents
can be determined in cell cultures or experimental animals by
standard pharmaceutical procedures for determining the LD.sub.50
(the dose lethal to 50% of the population) and the ED.sub.50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD.sub.50/ED.sub.50.
Agents that exhibit large therapeutic indices are preferred. When
an agent exhibits toxic side effects, a delivery system that
targets the agent to the site of affected tissue can be used to
minimize potential damage to non-CD70-expressing cells and,
thereby, reduce side effects.
[0153] Generally, the dosage of an antibody, derivative or ADC
administered to a patient with a CD70-expressing cancer is
typically 0.1 mg/kg to 100 mg/kg of the subject's body weight. More
typically, the dosage administered to a subject is 0.1 mg/kg to 10
mg/kg of the subject's body weight, even more typically 0.1 mg/kg
to 5 mg/kg, or 0.1 mg/kg to 3 mg/kg of the subject's body weight.
Generally, human antibodies have a longer half-life within the
human body than antibodies from other species due to the immune
response to the foreign proteins. Thus, lower dosages of ADCs
comprising humanized, chimeric or human antibodies and less
frequent administration is often possible.
[0154] Antibodies to CD70, derivatives and ADCs can also be
administered in combination with one or more other therapeutic
agents for the treatment or prophylaxis of pancreatic or ovarian
cancer. For example, combination therapy can include a second
cytostatic or cytotoxic agent (for example, an unconjugated
cytostatic or cytotoxic agent such as those conventionally used for
the treatment of cancers). Combination therapy can also include,
e.g., administration of an agent that targets a receptor or
receptor complex other than CD70 on the surface of CD70-expressing
cancer cells. Typically, such an antibody or ligand binds to a cell
surface receptor on CD70-expressing cancer cells and enhances the
cytotoxic or cytostatic effect of the anti-CD70 antibody by
delivering a cytostatic or cytotoxic signal to the CD70-expressing
cancer cells.
[0155] Other drugs that can administered with the agent s include
growth factor inhibitors, or anti-angiogenesis factors. For
example, a drug called erlotinib, an epidermal growth factor
receptor tyrosine kinase inhibitor, can be used to treat advanced
pancreatic cancer. (Bareschino et al., 2007, Ann Oncol. Suppl 6:
35-41.) Such combinatorial administration can have an additive or
synergistic effect on disease parameters (e.g., severity of a
symptom, the number of symptoms, or frequency of relapse).
[0156] The present methods can be combined with other means of
treatment such as surgery, radiation, targeted therapy,
immunotherapy, use of growth factor inhibitors, or
anti-angiogenesis factors.
[0157] Surgery is a preferred treatment and is frequently necessary
to obtain a tissue specimen for differential diagnosis via its
histology. Improved survival is attributed to more accurate staging
of the disease and a higher rate of aggressive surgical excision of
tumor in the abdomen. The type of surgery depends upon how
widespread the cancer is when diagnosed (the cancer stage), as well
as the presumed type and grade of cancer.
[0158] For patients suffering from pancreatic adenocarcinoma, the
surgeon can perform the Whipple procedure (also called,
pancreaticoduodenectomy). In this procedure, the head of the
pancreas and sometimes the body of the pancreas are removed along
with parts of the stomach and small intestine, the gallbladder,
part of the common bile duct, and some nearby lymph nodes. (See,
e.g., Michalski et al., 2007, Nat. Clin. Pract. Oncol.
4(9):526-35.) For patients suffering from endocrine tumors of the
pancreas (islet cell tumors), surgery is a viable option. (See,
e.g., Akerstrom and Hellman, 2007, Best Pract. Res. Clin.
Endocrinol. Metab. 21(1):87-109.)
[0159] In ovarian cancer patients, the surgeon can remove one
(unilateral oophorectomy) or both ovaries (bilateral oophorectomy),
the fallopian tubes (salpingectomy) and the uterus (hysterectomy).
For some very early tumors such as those in stage I, only the
involved ovary and fallopian tube will be removed ("unilateral
salpingo-oophorectomy," USO), especially in young females who wish
to preserve their fertility. In advanced malignancy, where complete
resection is not feasible, as much tumor as possible is removed
(debulking surgery). In cases where this type of surgery is
successful, the prognosis is improved compared to patients where
large tumor masses (more than 1 cm in diameter) are left behind.
Minimally invasive surgical techniques can facilitate the safe
removal of very large (greater than 10 cm) tumors with fewer
complications of surgery. (See, e.g., Ehrlich et al., 2007, J.
Pediatr. Surg. 42 (5): 890-3.)
[0160] Chemotherapy refers to the use of anti-cancer or cytotoxic
drugs to kill cancer cells. Chemotherapy can be given to the
patient before or after surgery. Depending on the histology of the
tumor, some kinds of tumor (particularly teratoma) are not
sensitive to chemotherapy. Intravenous chemotherapy with drugs such
as, e.g., gemcitabine, 5-flourouracil, cisplatin or mitomycin C can
be used to treat pancreatic cancer. Intravenous chemotherapy such
as gemcitabine, topotecan, doxorubicin, liposomal doxorubicin,
carboplatin, paclitaxel can be used to treat ovarian cancer.
Chemotherapy that is partly intravenous and partly intraperitoneal
can also improve median survival time. (The Chemotherapy Source
Book (3rd edition). Ed. Perry. Lippincott, Williams and Wilkins,
2001; Oxford Textbook of Palliative Medicine. (2nd Ed.) Derek Doyle
et al. Oxford University Press. 1999).
[0161] Radiation therapy is treatment with high energy rays, such
as x-rays, to kill or shrink cancer cells while doing as little
harm as possible to normal cells. Radiation can be given to the
patient before or after surgery. Radiation therapy can also be used
to treat pancreatic cancer which has not spread but cannot be
removed by surgery. Radiation therapy is not often used to treat
cancer of the ovary, but may occasionally be used, if appropriate.
Combination radiation and chemotherapy can be used for patients
whose tumors are too widespread to be removed by surgery.
[0162] An anti-CD70 antibody, derivative or ADC can be administered
concurrently to a patient undergoing surgery, chemotherapy or
radiation therapy treatments. Alternatively, a patient can undergo
surgery, chemotherapy or radiation therapy prior or subsequent to
administration of an anti-CD70 antibody, derivative or ADC by at
least an hour and up to several months, for example at least an
hour, five hours, 12 hours, a day, a week, a month, or three
months, prior or subsequent to administration of the ADC or ADC
derivative.
VII. Kits
[0163] The invention provides diagnostic kits for use with the
above detection methods. The kits typically contain an antibody or
fragment thereof that specifically binds to denatured CD70 useful
for detection as described above. One or more additional containers
may enclose elements, such as reagents or buffers, to be used in
the assay. Such kits can also, or alternatively, contain a
detection reagent that contains a reporter group suitable for
direct or indirect detection of antibody binding.
[0164] The invention further provides pharmaceutical kits for
treating pancreatic and ovarian cancers. Typically, such kits
contain reagents formulated as a therapeutic composition as
described herein, and can be in any of a variety of forms suitable
for distribution in a kit. Such forms can include a liquid, powder,
tablet, suspension and the like formulation for providing the
agents, such as anti-CD70 antibodies, derivatives or ADCs. The kits
can also include a pharmaceutically acceptable diluent (e.g.,
sterile water) for injection, reconstitution or dilution of the
lyophilized antibody, derivative or ADC.
[0165] The invention further provides combined kits for diagnosis
and therapy. Such kits typically include at least one antibody that
binds preferentially to denatured CD70 over native CD70 for use in
detection in fixed tissue sections and a different antibody that
binds to native CD70 at least as well if not better than denatured
CD70 for use in treatment.
[0166] Kits also typically contain a label or instructions for use
in the methods of detection and/or treatment described herein. The
label or instruction refers to any written or recorded material
that is attached to, or otherwise accompanies a kit at any time
during its manufacture, transport, sale or use. It can be a notice
in the form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. The label or instruction can also
encompass advertising leaflets and brochures, packaging materials,
instructions, audio or video cassettes, computer discs, as well as
writing imprinted directly on the pharmaceutical kits.
[0167] The invention is further described in the following
examples, which are not intended to limit the scope of the
invention. Cell lines described in the following examples were
maintained in culture according to the conditions specified by the
American Type Culture Collection (ATCC) or Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany
(DMSZ). Cell culture reagents were obtained from Invitrogen Corp.,
Carlsbad, Calif., or other suppliers.
EXAMPLES
Example 1
Preparation of Monoclonal Antibodies SG-21.1C1.B3 and
SG-21.5D12.C3
[0168] Monoclonal antibodies SG-21.1C1.B3 and SG-21.5D12.C3 were
produced using B-cells from spleen or lymph nodes removed from a
mouse that was challenged several times with the immunogen,
denatured extracellular domain of CD70 (CD70-ECD). These B-cells
were then fused with myeloma tumor cells to produced hybridomas.
Large numbers of monoclonal antibodies were thus produced from
these hybridomas. The hybridomas were diluted to ensure clonality
and grown.
[0169] Antibodies from the different clones were tested for their
ability to bind to the denatured CD70 antigen with a test such as
an ELISA using Flag-CD70 or a differential FMAT screen (Applied
Biosystems, Foster City, Calif.) using fixed 293F expressing
cynomolgus monkey ("cyno") CD70 and denatured L540cy cells, which
express CD70. Untransfected 293F and L540cy cells were used as a
negative control. (Some L540cy cells were positive for CD70).
[0170] The results showed that over 100 hybridomas tested positive
for their ability to bind to denatured CD70. Antibodies from two
positive hybridomas, SG-21.1C1.B3 ("1C1") and SG-21.5D12.C3
("5D12"), were selected for immunohistochemistry.
Example 2
Preparation of Formalin-Fixed Paraffin Embedded (FFPE) Samples
[0171] Formalin-fixed paraffin embedded tissues were prepared
according to standard methods, as described in fixed in Theory and
Practice of Histotechnology, Second Edition. 1980, Sheehan, D. C.
and Hrapchak, B. B., editors (Battelle Press (Columbus, Ohio).
Chapter 3, pp. 59-78).
[0172] The preparation of cells by FFPE was similar to that of the
tissue preparation. Briefly, cells were plated in suitable culture
media at about 15,000 cells/well 1 day prior to fixation. The cells
were fixed as follows: The cells washed 2.times. with PBS and then
fixed with 10% formalin at room temperature for 45 minutes.
Afterwards, the cells were washed 2.times. with PBS and then
permeablized with PBS+0.5% Triton X-100 at room temperature for 15
minutes. The cells were then washed 1.times. with PBS and stored in
PBS+0.02% sodium azide at 4.degree. C. Prior to FMAT screening, the
PBS+azide was removed and the plate was blocked with PBS+5% goat
serum at room temperature for 30 minutes.
Example 3
Preparation of Tissue Microarrays for Immunohistochemistry
[0173] Tissue microarrays of FFPE tissue sections were obtained
from commercial sources, including US Biomax, TriStar or Cybrdi.
Tissue microarray are also prepared according to the Yale Tissue
Microassay Construction Protocols, Version 1.0, and later
updates.
Example 4
Development of Monoclonal Antibodies SG-21.1C1.B3 and SG-21.5D12.C3
as Immunohistochemistry Reagents for FFPE Samples
[0174] A Immunohistochemical Testing of CD70 Clones
[0175] An expression construct encoding cynomolgus monkey CD70 was
made by cloning a full-length cynomolgus CD70 gene into an
expression vector. This construct was transfected into 293F cells.
These 293F:CD70 transfected cells served as the positive control
for staining with 1C1 and 5D12 antibodies. The parental 293F cell
line served as the negative control. For tissue staining, 786-O
cells, that express CD70, served as the positive control.
[0176] The cells and tissues were fixed and embedded in paraffin
wax according to the procedure described in Example 2. IHC staining
and antigen retrieval was performed using a Vision BioSystems
Bond-Max.TM. system (now Leica Microsystems). Antigen retrieval was
performed for 40 minutes using the EDTA retrieval method.
Alternatively, antigen retrieval was performed using the Trilogy
antigen retrieval system (Cell Marque, Hot Springs, Ark.) at a
temperature of 99-100.degree. C. for 1 hour.
[0177] Both the 1C1 primary antibody or the 5D12 primary antibody
stained strongly all fixed 293F:CD70 transfected cells, whereas no
staining was detected in the parental 293 cells. The results also
showed strong staining in the 786-O cells (a renal cell carcinoma)
whereas background staining was detected in a Ramos xenograft
control.
[0178] The 1C1 clone was further subcloned and a subclone, 1C1-B3
(SG-21.1C1.B3) was selected. Likewise, 5D12 was further subcloned
and a subclone, 5D12-C3 (SG-21.5D12.C3) was selected. These two
subclones were purified and used as primary antibodies to stain
786-O xenograft and Ramos xenograft samples. The results showed
that subcloning and purification of 1C1-B3 and 5D12-C3 removed
background staining in the Ramos xenograft.
[0179] Hybridomas producing the anti-CD70 antibodies have been
deposited with the American Type Culture Collection (ATCC) at 10801
University Boulevard, Manassas, Va. 20110-2209. The cell line
designated SG-21.1C1.B3, producing the antibody 1C1 having the ATCC
accession number PTA-8733 has been deposited on Oct. 24, 2007 at
the ATCC; the cell line designated SG-21.5D12.C3 producing the
antibody 5D12 having the ATCC accession number PTA-8734 has been
deposited on Oct. 24, 2007 at the ATCC.
[0180] B. Western Blotting with SG-21 Antibodies 1C1 and 5D12
Compared with 2B3 Antibody
[0181] To determine whether SG-21 antibodies 1C1 and 5D12
antibodies detect CD70 in cell and tissue lysates, Western blots
experiments were conducted. Another CD70 binding antibody, 2B3, was
also used for comparison. Membrane preparations were prepared from
786-O cells, 293F:cynoCD70 (expressing cyno CD70) cells and 293F
cells as a negative control. To prepare membrane extracts, cell
were lysed in hypotonic buffer solution and centrifuged at
10,000.times.g for 10 minutes at 4.degree. C. to remove debris. The
supernatants were then centrifuged at 100,000.times.g for 30
minutes at 4.degree. C. to pellet the membrane fractions. The
membrane fractions (pellet) were dissolved in 0.5% NP40 in 50 mM
Tris plus 150 mM NaCl and 5 mM EDTA. All sample preparation was
done in the presence of protease inhibitors to keep CD70 intact.
The quantity of the proteins was measured at 570 nm using a BCA Kit
(Pierce). CD70 ECD (extracellular domain of CD70) and Flag tagged
CD70-ECD were also prepared by standard methods. The protein
samples were denatured at 90.degree. C. for 3 minutes and chilled
on ice for 3 minutes. The samples were separated using SDS-PAGE and
then transferred to a nitrocellulose membrane for detection. Four
identical SDS-PAGE gels and membranes were prepared. The membranes
were blocked in PBS with 1% BSA and 2% non-fat milk with 0.05%
Tween (polysorbate). Each primary antibody was then added to the
solution at 0.5 .mu.g/ml and allowed to incubate for 4 hours at
room temperature in PBS with 0.05% Tween. The membranes were washed
and a secondary antibody-enzyme conjugate, which recognized the
primary antibody was added and incubated for 45 minutes at room
temperature. The membranes were washed and incubated with a
chemiluminescent substrate to detect CD70.
[0182] Referring to FIG. 1, the results showed that SG-21
antibodies 1C1 and 5D12 antibodies detected CD70 in the 786-O and
the 293F:CD70 transfectants, but not in the negative control 293F
cells. Notably, the detected bands were of identical size in the
786-O and 293F:CD70 transfectants. SG-21 antibody 2B3 did not
detect CD70 in 786-O and 293:cynoCD70 samples but detected CD70ECD
and flag-CD70-ECD. The result indicated that 1C1 and 5D12
antibodies detected CD70 in the samples.
[0183] C. CD70 Expression in Tumor and Non-Tumor Cell Lines Using
SG21.1C1 Antibody
[0184] Tissue microarrays were obtained from commercial sources or
custom prepared that contained samples of the following cancers:
Breast Carcinoma, Ovarian Carcinoma, Mesothelioma, Osteosarcoma,
Prostate Carcinoma, Hepatocellular Carcinoma, Glioblastoma,
Anaplastic Astrocytonma, Uterine Cancer, Embryonic Cancer,
Epidermoid Carcinoma, Multiple Myeloma, Hodgkin Lymphoma, Non-T,
Non-B All Histiocytic Lymphoma, Non-Hodgkin Lymphoma (NHL) Burkitt
Lymphoma, NHL follicular Lymphoma, Acute Myeloid Leukemia (AML),
Anaplastic Large Cell Lymphoma (ALCL), Erythroleukemia, Colon
Carinoma, Non-small Cell Lung Cancer (NSCLC), Small Cell Lung
Cancer (SCLC), Renal Cell Carninoma (RCC), RCC clear cell type, RCC
Papillary type, Melanoma, Pancreatic Carcinoma and Bladder
Carcinoma. The following cells or cell lines were also used:
Epstein-Barr virus-transformed B lymphoblastoid cell line
(EBV-LCL), normal human mammary epithelial cells (HMEC), normal
human vascular endothelial cells (HUVEC), normal blood mononuclear
(PBMC), normal human aortic endothelial cells (HAEC), normal human
renal endothelial cells (HREC), normal human Lung Microvascular
Endothelial Cells (HMVEC-L), normal human endo-neonatal dermal
microvascular endothelial cells (HMVEC-neo) and normal human
pulmonary artery endothelial cells (HPAEC).
[0185] The tissue microarrays were immuno-stained with SG21.1C1
antibodies using the protocol described above. Staining was
observed in the following cell lines: Ovarian Carcinoma cell line
SK-OV-3; Glioblastoma cell line GMS-10; Multiple Myeloma cell lines
LB, LP-1, AMO-1, I-310 (MM.1R), C2E3 (MM.1S), MOLP-8, JJN-3 and
L363; Hodgkin lymphoma cell lines KMH2, HS445, RPMI-1666, L248 and
HD-M-YZ; EBV-LCL WIL2-S, Farage and IM-9; NHL, follicular cell line
WSU-NHL; RCC clear cell type cell lines Caki-1, Caki-2, 786-O and
769-P; RCC papillary type cell lines CAL54, A498; RCC SK-RC-6 and
SK-RC-7; Melanoma cell lines A375M and A375SM, Pancreatic Carcinoma
cell line PANC-1 and Bladder Carcinoma T24. The staining of some
cell lines not identified was faint or mixed. Some cell lines have
cytoplasmic staining. For the staining of pancreatic and ovarian
cell lines, see FIGS. 2 and 3, respectively.
[0186] D. Comparative Staining of 1C1 and 5D12 Antibodies
[0187] 1C1 and 5D12 antibodies were compared by staining 293F,
293F-cynoCF0 cell lines, RCC Caki-1, normal tonsil, skeletal
muscle, bladder, lymphoid tissues (lymph node, thymus, spleen) from
normal Cynomolgus monkeys, normal human thymus, kidney and skeletal
muscle tissues and pancreatic tumor tissues. The immuno-staining
protocol was as described above.
[0188] The results showed that 1C1 and 5D12 similarly stained 293F
and 293-cyno CD70 cell lines (data not shown); RCC Caki-1 and
cynomolgus tonsil tissues (data not shown); lymphoid tissues (lymph
node, thymus, spleen) from normal Cynomologous monkeys (data not
shown) and pancreatic tumor tissues (see FIG. 4). Some differential
staining of 1C1 and 5D12 was observed in normal human and monkey
skeletal muscle tissues and normal monkey bladder tissues in which
5D12 stained the tissues but 1C1 did not. Differential staining was
also observed in normal human thymus and kidney tissues in which
5D12 stained cytoplasmically.
Example 5
CD70 Expression in Normal Colon and Colorectal Cancer Tissues and
Normal Pancreas and Pancreatic Cancer Tissues with 1C1 Antibody
[0189] Before assessing the whole panel of tumor tissues from
different cancer types, CD70 expression was first assayed in colon
and pancreatic cancers. The immuno-staining protocol was as
described in Example 4. The chromagen used for these studies was
Fast Red.
[0190] The results showed that in normal colon tissues, only rare
lymphocytes were stained positive for CD70. Other cells were
negative for CD70. In one sample of colon cancer tissue, the tumor
cells were stained positive. In a second sample of colon tissue,
the tumor cells were negative but the stroma was positive.
Likewise, in normal pancreatic tissues, the staining was negative
for CD70. In one sample of pancreatic cancer tissues, tumor cells
were positive and in a second sample, tumor cells were negative but
the stroma was positive. (See FIG. 5).
Example 6
CD70 Expression in Tumor Tissues with SG21.1C1 Antibody
[0191] Tumor tissues were obtained from the following tumor types:
Hodgkin lymphoma, kidney, lymphoma, multiple myeloma, pancreas,
larnyx/pharynx, ovary, colon and breast. The normal and tumor
tissues were prepared according to the tissue array protocol
described in Example 3 and the immuno-staining protocol was as
described in Example 4A. 1C1 antibody was used as the primary
antibody. The immunohistochemical (IHC) expression was later
evaluated based on staining intensity and percentage of tumor
involved. Staining intensity was ranked from 1 to 4, with 1
indicating minimal staining; 2, mild staining; 3, moderate staining
and 4, strong staining. Percentage of tumor involved was also
ranked from 1 to 4, with 1 indicating 0-5%; 2, 5-25%; 3, 25-75% and
4, 75%-100%. The measurements were qualitative. For Hodgkin
lymphoma, IHC expression of CD70-positive cells was assayed based
on Reed-Sternberg cell evaluation. Reed-Sternberg cells are large
cells of unknown origin, usually multinucleate, whose presence is
the common histological characteristic of Hodgkin lymphoma.
[0192] Hodgkin lymphoma tumor tissues had the highest percentage
(97% or 33/34) of CF70-positive tumor cells over cells in the total
tumors. Kidney tumor had the second highest percentage (70% or
14/20) of CF70-positive tumor cells over cells in the total tumors.
Lymphoma had the third highest percentage (61% or 72/119) of
CD70-positive tumor cells over cells in the total tumors. Multiple
Myeloma had the fourth highest percentage (42% or 13/31) of
CF70-positive tumor cells over cells in the total tumors. Pancreas
cancer had the fifth highest percentage (25% or 35/140) of
CF70-positive tumor cells over cells in the total tumors.
[0193] FIG. 6 illustrates a graph of staining intensity and % of
tumor staining for 35 of 140 pancreatic samples giving a positive
signal for CD70. The figure shows a complex relationship between
the staining intensity and percentage of tumor staining. That is,
some tumors have a high percentage of cells staining but at low
intensity, others have a low percentage of cells staining but at
high intensity, and others show intermediate staining intensity and
percentage cells staining. Larnyx/pharnyx cancer had the sixth
highest percentage (22% or 18/82) of CF70 positive-tumor cells over
cells in the total tumors. Ovarian Cancer had the seventh highest
percentage (15% or 37/241) of CF70-positive tumor cells over cells
in the total tumors.
[0194] FIG. 7 illustrates staining intensity and percent of tumor
staining for 37 of 241 CD70 staining ovarian cancers. There was
some association between staining intensity and percentage of cells
staining. Colorectal Cancer had the seventh highest percentage (9%
or 17/194) of CF70-positive tumor cells over cells in the total
tumors. Breast Cancer had the lowest percentage (2% or 5/204) of
CF70-positive tumor cells over cells in the total tumors.
[0195] To summarize, based on CD70-positive tumors/total tumors,
stain intensity (cellular target expression), percentage of
CD70-positive tumor involvement (tumor target expression), general
indication ranking of the tumor types is as follows:
Hodgkin>Kidney>Lymphoma>Multiple
Myeloma>Pancreatic>Larnyx/pharnyx>Ovarian>Colorectal>Breas-
t. The results of all the tumor tissues are shown on Table 1.
TABLE-US-00001 TABLE 1 CD70 Expression in Various Cancers CD70+/
Tumor Type Total % of CD70+ Hodgkin 33/34 97 Kidney 204/283 72
Lymphoma 72/119 61 Multiple 13/31 42 Myeloma Pancreas 35/140 25
Larnyx/ 18/82 22 Pharynx Ovary 37/241 15 Skin 4/30 13 Lung (all)
40/475 8 Lung 17/172 10 adenocarcinoma Colon 17/194 9 Breast 5/204
2
Example 7
h1F6-Drug Conjugates Show Efficacy in an Ovarian Carcinoma Cell
Line
[0196] To confirm the efficacy of known anti-CD70 antibody drug
conjugates against representative cell lines for these new cancers,
SKOV-3 cells were prepared and tested as generally described
previously for other cell lines. (See International Patent
Publication WO 2006-113909.) The cells were incubated with the
following anti-CD70 antibody drug conjugates: h1F6-vc-MMAF(4),
h1F6vc-MMAE(4), 1F6mc-MMAF(4), or h1F6mc-MMAF(8) or free MMAF. (See
the specification and U.S. Patent Application Publication Nos.
2005-0238649 and 2006-0233794 for a description of the drug
linkers. The number in parentheses after each conjugate indicates
the average drug loading per antibody.) The SKOV-3 cells were
incubated with the conjugates at the indicated concentrations for
96 hours. For these studies, viability was determined using Promega
CelltiterGlo.
TABLE-US-00002 TABLE 2 h1F6vc- h1F6vc- h1F6mc- h1F6mc- MMAF(4)
MMAE(4) MMAF(4) MMAF(8) MMAF 5 ng/ml 60 ng/ml 29 ng/ml 12 ng/ml 18
nM
[0197] Referring to FIG. 8, all four anti-CD70 ADCs were active
against this ovarian cancer cell line. Referring to Table 2, the
IC50's are shown. These IC50's are consistent with those reported
for other CD70-expressing cancer cell lines. These results confirm
that anti-CD70 ADCs bound to CD70 on this cancer cell line is
internalized and releases the auristatin payload.
Example 8
h1F6-Drug Conjugates Show Efficacy in a CD70 Transfected Pancreatic
Carcinoma Cell Line
[0198] To confirm the efficacy of known anti-CD70 antibody drug
conjugates against representative cell lines, the pancreatic cell
lines HPAFII, PANC-1 and MiaPaCa-2 were transfected with a modified
cynomolgus CD70 encoding nucleic acid. MiaPaCa-2 cells do not
express detectable levels of CD70 protein. h1F6 binds to native
CD70 protein expressed by these transfected cell lines. The
expression of CD70 by the transfected cell lines was confirmed by
FACS analysis (data not shown). The activity of h1F6 mc-MMAF(4)
(SGN-75) was tested on the transfected pancreatic cell lines
generally described previously for other cell lines. (See Example 7
an International Patent Publication WO 2006-113909.) (The number in
parentheses after the conjugate indicates the average drug loading
per antibody.) Referring to FIGS. 9A-C, the cells were incubated
with the conjugate at the indicated concentrations for 96 hours.
Referring to FIG. 9A, the activity of the conjugate on the
transfected HPAFII cells is shown. Cell viability was determined
using Promega CelltiterGlo. Referring to FIGS. 9B and 9C, the
activity of the conjugate on transfected PANC-1 and MiaPACa-2
transfected cell lines is shown. For these studies, cell viability
was determined using rezasurin, as described previously. The PANC-1
Activity of Promega CelltiterGlo. The conjugate showed cytotoxic
activity on all these cell lines in vitro.
Example 9
h1F6-Drug Conjugates Show Efficacy in a Xenograft Model of
Pancreatic Cancer
[0199] Nude (nu/nu) female mice (7 animals/group) were implanted
with CD70-transfected MiaPaCa tumor chunks (prepared as described
in Example 8) via trocar into the right lateral flank. Dosing with
either SGN-75 or nonbinding control ADC (3 mg/kg) started when
tumors reached 100 mm.sup.3 (q4d.times.4 ip). Tumor volumes were
monitored and animals were euthanized when tumor volume reached
1000 mm.sup.3. Referring to FIG. 10, the data were plotted in 2
ways: FIG. 10A: Median tumor volume plots were continued for each
group until one or more animals were euthanized. FIG. 10B:
Kaplan-Meier curve shows time for tumor to reach 800 mm.sup.3 for
individual animals in each group. Treatment with SGN-75 was
effective in this xenograft model.
[0200] The present invention is not limited in scope by the
specific embodiments described herein. Various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications are intended to fall
within the scope of the appended claims. Unless otherwise apparent
from the context any step, element, embodiment, feature or aspect
of the invention can be used in combination with any other. All
patent filings, and scientific publications, accession numbers and
the like referred to in this application are hereby incorporated by
reference in their entirety for all purposes to the same extent as
if so individually denoted.
Sequence CWU 1
1
41137PRTMus_musculus 1Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met
Ala Ala Ala Gln Ser 1 5 10 15 Ala Gly Ala Gln Ile Gln Leu Val Gln
Ser Gly Pro Glu Val Lys Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Gly Met
Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60 Lys Trp Met
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 Asp
Ala Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90
95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr
100 105 110 Tyr Phe Cys Ala Arg Asp Tyr Gly Asp Tyr Gly Met Asp Tyr
Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser 130 135
2132PRTMus_musculus 2Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Ala 20 25 30 Val Ser Leu Gly Gln Arg Ala
Thr Ile Ser Cys Arg Ala Ser Lys Ser 35 40 45 Val Ser Thr Ser Gly
Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro
Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly
Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90
95 Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
100 105 110 Gln His Ser Arg Glu Val Pro Trp Thr Phe Gly Gly Gly Thr
Lys Leu 115 120 125 Glu Ile Lys Arg 130 3137PRTMus_musculus 3Met
Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Asp 1 5 10
15 Val Gln Ser Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Met Thr
20 25 30 Pro Gly Ala Ser Val Thr Met Ser Cys Lys Thr Ser Gly Tyr
Thr Phe 35 40 45 Ser Thr Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro
Gly His Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Leu Gly Pro Ser
Gly Tyr Thr Asp Tyr Asn 65 70 75 80 Glu Lys Phe Lys Ala Lys Ala Thr
Phe Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu
Ser Ser Leu Ala Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala
Arg Trp Asp Arg Leu Tyr Ala Met Asp Tyr Trp Gly 115 120 125 Gly Gly
Thr Ser Val Thr Val Ser Ser 130 135 4132PRTMus musculus 4Met Glu
Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15
Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Thr 20
25 30 Val Ser Leu Gly Gln Lys Thr Thr Ile Ser Cys Arg Ala Ser Lys
Ser 35 40 45 Val Ser Thr Ser Gly Tyr Ser Phe Met His Trp Tyr Gln
Leu Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Leu Ala
Ser Asp Leu Pro Ser 65 70 75 80 Gly Val Pro Ala Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile His Pro Val Glu
Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln His Ser Arg Glu
Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Thr
Arg 130
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