U.S. patent application number 11/709917 was filed with the patent office on 2007-09-06 for methods and compounds for lymphoma cell detection and isolation.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Tomoyuki Endo, Tetsuya Fukuda, Thomas J. Kipps.
Application Number | 20070207510 11/709917 |
Document ID | / |
Family ID | 37968632 |
Filed Date | 2007-09-06 |
United States Patent
Application |
20070207510 |
Kind Code |
A1 |
Kipps; Thomas J. ; et
al. |
September 6, 2007 |
Methods and compounds for lymphoma cell detection and isolation
Abstract
Compositions comprising a purified, isolated antibody, humanized
antibodies and precipitates directed against ROR-1, wherein the
antibody binds ROR-1 with moderate to high affinity. The
compositions may be used for detecting and isolating an amount of
ROR-1 in a subject sample, and to evaluate the appearance, status,
course, or treatment of a ROR-1 cancer in a subject. The ROR-1
antibodies are especially useful in identifying lymphomas and
ademocarcinomas. Vaccines and related methods for protecting a
subject against diseases that involve expression of ROR-1 are also
provided.
Inventors: |
Kipps; Thomas J.; (San
Diego, CA) ; Fukuda; Tetsuya; (Kanagawa, JP) ;
Endo; Tomoyuki; (Hokkaido, JP) |
Correspondence
Address: |
DLA PIPER US LLP
4365 EXECUTIVE DRIVE
SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
|
Family ID: |
37968632 |
Appl. No.: |
11/709917 |
Filed: |
February 21, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US06/42689 |
Oct 30, 2006 |
|
|
|
11709917 |
Feb 21, 2007 |
|
|
|
60731210 |
Oct 28, 2005 |
|
|
|
Current U.S.
Class: |
435/7.23 ;
530/388.8 |
Current CPC
Class: |
A61P 3/00 20180101; G01N
33/57415 20130101; G01N 33/57426 20130101; C07K 16/081 20130101;
G01N 33/57419 20130101; A61K 39/0011 20130101; A61K 2039/53
20130101; C07K 16/3061 20130101; A61K 39/00118 20180801 |
Class at
Publication: |
435/007.23 ;
530/388.8 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07K 16/30 20060101 C07K016/30 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made in part with Government support
under National Institutes of Health Grant 5P01 CA81543. The
Government has certain rights in the invention.
Claims
1. A composition comprising a purified, isolated antibody directed
against ROR-1, wherein the antibody binds ROR-1 with moderate to
high affinity.
2. A composition according to claim 1, wherein the antibody is an
anti-ROR-1 polyclonal antibody, a monoclonal antibody, or a
functional antibody fragment.
3. A composition according to claim 2, wherein the antibody
comprises a sequence expressed by a heavy chain sequence and at
least one light chain sequence of SEQ ID NOs: 1-5.
4. A composition according to claim 2, wherein the antibody is a
polyclonal antibody.
5. A composition according to claim 2, wherein the antibody is a
monoclonal antibody.
6. A composition according to claim 2, wherein the antibody is a
functional antibody fragment.
7. A composition according to claim 1, wherein the antibody is
selected from the group consisting of whole antibody, humanized
antibody, chimeric antibody, Fab fragment, Fab' fragment,
F(ab').sub.2 fragment, single chain Fv fragment and diabody.
8. A composition according to claim 1, wherein the antibody has an
affinity to binding ROR-1 with a dissociation constant of below a
Kd value selected from the group consisting of 10.sup.-6 mol/l,
10.sup.-7 mol/l, and 10.sup.-8 mol/l.
9. A composition according to claim 1, wherein the antibody is a
detectably labeled antibody.
10. A composition according to claim 1, further comprising a
pharmaceutically acceptable agent.
11. A method for detecting an amount of ROR-1 in a subject sample,
the method comprising: (a) contacting the subject sample with the
anti-ROR-1 antibody of claim 1; and (b) detecting immunoreactivity
between the anti-ROR-1 antibody and ROR-1 in the sample.
12. A method according to claim 11, wherein the antibody is
covalently attached to a detectable label.
13. A method according to claim 11, wherein the immunoreactivity
detection is provided by immunoperoxidase staining,
immunofluorescence, immunoelectronmicroscopy, or ELISA.
14. A method according to claim 11, further comprising (c)
correlating binding of the antibody to a standardized antibody
binding profile, wherein such correlation provides quantitative
value for total CPR in the sample.
15. A method for detecting a ROR-1 cancer, the method comprising:
detecting the presence or quantity of ROR-1 protein in a subject
sample.
16. A method according to claim 15, wherein the ROR-1 cancer is a
lymphoma or adeno carcinoma.
17. A method according to claim 16, wherein the cancer is selected
from the group consisting of CLL, small lymphocytic lymphoma,
marginal cell B-Cell lymphoma, Burkett's Lymphoma, colon
adenocarcinoma, colorectal adenocarcinoma, and breast
adenocarcinoma.
18. A method according to claim 15, wherein detection of ROR-1
comprises the method of claim 10.
19. A method for treating a ROR-1 cancer in a subject, the method
comprising: administering to the subject in need thereof a
therapeutically effective amount of a ROR-1 receptor agonist.
20. A method according to claim 19, wherein the ROR-1 cancer is a
lymphoma or adenocarcinoma.
21. A method according to claim 19, wherein the lymphoma is
selected from the group consisting of CLL, small lymphocytic
lymphoma, marginal cell B-Cell lymphoma, and Burkett's Lymphoma,
colon adenocarcinoma, and breast adenocarcinoma.
22. A method according to claim 19, wherein the ROR-1 receptor
agonist is the anti-ROR-1 antibody of claim 1.
23. A method according to claim 22, wherein the antibody is
administered in an amount of (i) about 0.05 mg to about 2.5 mg;
(ii) about 0.1 mg to about 1 mg; or (iii) about 0.3 mg to about 0.5
mg.
24. A method according to claim 22, wherein the anti-ROR-1 antibody
is a polyclonal antibody, a monoclonal antibody, or a functional
antibody fragment.
25. A method according to claim 22, wherein the anti-ROR-1 antibody
is selected from the group consisting of whole antibody, humanized
antibody, chimeric antibody, Fab fragment, Fab' fragment,
F(ab').sub.2 fragment, single chain Fv fragment and diabody.
26. A method according to claim 19, wherein the ROR-1 receptor
agonist is an antisense inhibitor of ROR-1.
27. A method according to claim 26, wherein the ROR-1 antisense
inhibitor is administered in an amount of (i) about 10 .mu.g/day to
about 3 mg/day; (ii) about 30 .mu.g/day to about 300 .mu.g/day; or
(iii) about 100 .mu.g/day.
28. A method according to claim 19, wherein the ROR-1 receptor
agonist is administered by injection, inhalation, orally, liposome,
or retroviral vector.
29. A diagnostic method for evaluating the appearance, status,
course, or treatment of a ROR-1 cancer in a subject, the method
comprising: (a) contacting a subject sample with the anti-ROR-1
antibody of claim 1; and (b) detecting immunoreactivity between the
anti-ROR-1 antibody and ROR-1 to determine presence or quantity of
ROR-1 in the sample.
30. A method according to claim 29, wherein the antibody
specifically binds to ROR-1.
31. A method according to claim 29, wherein a diagnostic criterion
or value is determined based on an increase or decrease in an
amount of ROR-1 in the subject compared to a control level(s) of
ROR-1 in a normal subject or sample.
32. A method according to claim 29, wherein immunoreactivity
detection is provided by immunoperoxidase staining,
immunofluorescence, immunoelectronmicroscopy, or ELISA.
33. A method according to claim 29, wherein the antibody is an
anti-ROR-1 polyclonal antibody, a monoclonal antibody, or a
functional antibody fragment.
34. A method according to claim 33, wherein the antibody is
selected from the group consisting of whole antibody, humanized
antibody, chimeric antibody, Fab fragment, Fab' fragment,
F(ab').sub.2 fragment, single chain Fv fragment and diabody.
35. A kit to detect the presence of ROR-1 protein comprising the
antibody of claim 1.
36. A vaccine composition comprising a polynucleotide encoding
ROR-1 protein or a fragment or variant thereof, and a
pharmaceutically acceptable carrier or diluent.
37. A vaccine composition comprising ROR-1 protein or a fragment or
variant thereof, and a pharmaceutically acceptable carrier or
diluent.
38. A method for protecting against the occurrence of diseases
involving expression of ROR-1 in a subject, the method comprising:
administering to the subject in need thereof a polynucleotide
encoding ROR-1 protein or a fragment or variant thereof in an
amount effective to induce a protective or therapeutic immune
response against ROR-1 in the subject, and a pharmaceutically
acceptable carrier or diluent.
39. A method for protecting against the occurrence of diseases
involving expression of ROR-1 in a subject, the method comprising:
administering to the subject in need thereof a ROR-1 protein or a
fragment or variant thereof in an amount effective to induce a
protective or therapeutic immune response against ROR-1 in the
subject, and a pharmaceutically acceptable carrier or diluent.
40. A method for identifying or isolating ROR-1 protein in a
sample, the method comprising: (a) contacting the sample with the
anti-ROR-1 antibody of claim 1; and (b) detecting immunoreactivity
between the anti-ROR-1 antibody and ROR-1 to determine presence or
quantity of ROR-1 in the sample.
41. A method according to claim 40, wherein the ROR-1 antibody is
conjugated to a magnetic bead.
42. A method for detecting minimal residual disease following
treatment of a ROR-1 cancer, the method comprising: (a) contacting
the sample with the anti-ROR-1 antibody of claim 1; and (b)
detecting immunoreactivity between the anti-ROR-1 antibody and
ROR-1 to determine presence or quantity of ROR-1 in the sample.
43. A method according to claim 42, wherein the ROR-1 antibody is
conjugated to a magnetic bead.
44. A humanized ROR-1 antibody.
45. A precipitate comprising a ROR-1 antibody and a polypeptide
selected from the group consisting of a ROR-1 protein, ROR-1
polypeptide fragment and ROR-1 polypeptide variant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional Patent
Application No. 60/731,210, filed Oct. 28, 2005, and to the PCT
application filed on Oct. 30, 2006, of which this is a
continuation-in-part.
FIELD OF THE INVENTION
[0003] The present invention generally relates to antibodies
directed against antigens specific for chronic lymphocytic
leukemia.
BACKGROUND
[0004] ROR-1 is an embryonic protein that is expressed uniquely on
certain cancer cells, including in chronic lymphocytic reukemia
(CLL), small lymphocytic lymphoma, marginal cell B-Cell lymphoma,
Burkett's Lymphoma, and other cancers (e.g., breast cancers), but
not on normal adult tissues and cells. Anti-ROR-1 antibodies raised
against ROR-1 peptide are commercially available, but monoclonal
anti-ROR-1 antibodies that react with the native ROR-1 protein have
not been made or isolated. In addition, no anti-ROR-1 antibodies
capable of detecting cell-surface expression of ROR-1 for flow
cytometric analysis have been made or isolated. What is needed,
therefore, is an antibody that can react with native ROR-1
protein.
SUMMARY OF THE INVENTION
[0005] Among the various aspects of the present invention is the
provision of an antibody directed to a surface receptor tyrosine
kinase protein expressed on cells found in samples of subjects with
a cancer, including lymphomas, CLL, small lymphocytic lymphoma,
marginal cell B-Cell lymphoma, Burkett's Lymphoma, renal cell
carcinoma, colon cancer, colorectal cancer, and breast cancer, but
not in blood or splenic lymphocytes of nonleukemic patients or
normal adults.
[0006] Briefly, therefore, the present invention is directed to an
antibody useful for differentiation between ROR-1 expressing cancer
cells ("ROR-1 cancer") and normal cells as well as immunotherapy
against ROR-1 cancers and determination of response to cancer
therapy.
[0007] The present invention includes compositions that include a
purified, isolated antibody that binds specifically to ROR-1
receptor protein.
[0008] The present invention includes methods for an immunoassay
that detects ROR-1 in a sample from a subject by contacting the
sample with a ROR-1-specific antibody and detecting
immunoreactivity between the antibody and ROR-1 in the sample.
[0009] In accordance with a further aspect of the invention, a
ROR-1 cancer is diagnosed in a subject by detecting the presence or
quantity of ROR-1 protein in a sample derived from the subject.
[0010] In accordance with yet another aspect of the invention, a
ROR-1 cancer is treated in a subject by administering to the
subject in need of such therapy a therapeutically effective amount
of a ROR-1 receptor agonist.
[0011] In accordance with yet another aspect, the appearance,
status, course, or treatment of a ROR-1 cancer in a subject is
evaluated by contacting a biological sample obtained from the
subject with an anti-ROR-1 antibody and detecting immunoreactivity
between the antibody and ROR-1 to determine presence or quantity of
ROR-1 in the sample.
[0012] In accordance with yet another aspect, also provided is a
vaccine composition comprising a polynucleotide encoding ROR-1
protein or a fragment or variant thereof, and a pharmaceutically
acceptable carrier or diluent.
[0013] In accordance with yet another aspect, also provided is a
vaccine composition comprising ROR-1 protein or a fragment or
variant thereof, and a pharmaceutically acceptable carrier or
diluent.
[0014] In accordance with yet another aspect, also provided is a
method for protecting against the occurrence of diseases involving
expression of ROR-1 in a subject, the method comprising
administering to the subject in need thereof a polynucleotide
encoding ROR-1 protein or a fragment or variant thereof in an
amount effective to induce a protective or therapeutic immune
response against ROR-1, and a pharmaceutically acceptable carrier
or diluent.
[0015] In accordance with yet another aspect, also provided is a
method for protecting against the occurrence of diseases involving
expression of ROR-1 in a subject, the method comprising
administering to the subject in need thereof ROR-1 protein or a
fragment or variant thereof in an amount effective to induce a
protective or therapeutic immune response against ROR-1 in the
subject, and a pharmaceutically acceptable carrier or diluent.
[0016] In accordance with yet another aspect, a humanized ROR-1
antibody is provided. In another aspect, a precipitate comprising a
ROR-1 antibody bound with a ROR-1 protein, fragment or variant is
provided. The ROR-1 antibody can be conjugated to a magnetic
bead.
[0017] Other objects and features will be in part apparent and in
part pointed out hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Those of skill in the art will understand that the drawings,
described below, are for illustrative purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0019] FIG. 1 shows change of serum antibody after Ad-CD154
therapy.
[0020] FIG. 1A is a series of scatter and line plots showing total
levels of IgG, IgA, and IgM. IgG, IgA, IgM blood concentrations,
measured just prior to initiating Ad-CD154 therapy (PRE) and 2-4
week following the final treatment time point (POST). The dashed
bar in each line graph indicates the minimum normal Ig
concentration. The concentration range of normal Ig levels is shown
to the left of legend.
[0021] FIG. 1B is a series of scatter and line plots showing
antibody response to recombinant Ad-CD154. Anti-adenovirus
antibodies were analyzed by an ELISA assay. Serial dilutions of
patient serum before (dotted line) and after (filled line)
treatment were incubated in 96 well plates coated with Ad-CD154.
Bound adenovirus-specific antibody was then detected using
AP-conjugated antibody specific for human Ig.
[0022] FIG. 1C is a series of bar graphs showing change of antibody
response against Adenovirus in serial samples. Anti-adenovirus
antibodies were analyzed by an ELISA using anti-isotype specific
secondary antibodies conjugated AP. The bar graphs represent the
mean increase in adenovirus-specific antibody over the baseline
pre-treatment antibody levels. IgE and IgG4 levels for all patients
were below the assay detection limits (data not shown).
[0023] FIG. 1D is a series of scatter and line plots showing
anti-tetanus-toxin antibody response before and after Ad-CD154
treatment. ELISA assay was performed with purified tetanus toxin
and sera from patients. Bound tetanus-specific antibody was
detected using AP conjugated goat anti-human Ig antibody.
[0024] FIG. 2 is a series of histograms showing antibody production
against surface molecules on CLL B cells by Ad-CD154 therapy.
Antibody bound on CD19+ CD3-cells were detected by goat anti-human
antibody.
[0025] FIG. 2A is a series of histograms showing diluted serum from
patient before (open histograms) or after (shaded histograms)
treatment was incubated with PBMC from CLL patient.
[0026] FIG. 2 B is a series of histograms showing diluted serum
from patient before (open histograms) or after (shaded histograms)
treatment was incubated with PBMC from a healthy donor.
[0027] FIG. 3 is an immunoblot of Immune Precipitates of Lysates
with 4A5 Probed With Rabbit anti-ROR-1 Raised Against Ror1
Peptides.
[0028] FIG. 4 is a series of images depicting gels that show
expression of ROR-1 in CLL B cells.
[0029] FIG. 4A are gel images of an immunoblot analysis of ROR-1
protein. Total cell lysates of PBMC from CLL patients or healthy
donor and those of splenocytes from CLL patients or idiopathic
thrombocytopenia purpura patient were analyzed by immunoblot using
rabbit anti-ROR-1 antibody (Cell signaling).
[0030] FIG. 4B are gel images showing ROR-1 expression in B cell
lines. Immunoblot analysis of total cell lysates of B cell lines
was performed.
[0031] FIG. 4C shows production of mouse anti-ROR-1 sera. CHO cells
stained with PKH26 and were mixed with CHO transfected ROR-1 cDNA
(CHO-ROR-1). Sera collected from mice before and after immunization
with ROR-1 cDNA were incubated with mixed CHO cells. Bound
antibodies were detected flow cytometry.
[0032] FIG. 4D is a series of histograms showing flow cytometric
analysis of expression of ROR-1 on cell surface of CLL. PBMC from
CLL patients and healthy donor were incubated antisera before (open
histograms) and after (shaded histograms) DNA immunization.
[0033] FIG. 5 is a series of histograms showing production of
anti-ROR-1 antibody detected by flow cytometric analysis.
[0034] FIG. 5A is a series of histograms where CHO (open
histograms) or CHO-ROR-1 (shaded histograms) was incubated with
serum from patients before (pre) or after (post) therapy.
Histograms indicated the bound human Ig detected by PE labeled goat
anti-human Ig.
[0035] FIG. 5 B shows results where CHO stained with PKH26 were
mixed and incubated with serum from patient. APC conjugated
anti-human Ig antibody was used for detection.
[0036] FIG. 6 shows production of anti-ROR-1 antibody detected by
ELISA.
[0037] FIG. 6A is a series of gel images showing production of
recombinant ROR-1 protein. ROR-1 extracellular region was fused
with rabbit IgG Fc region in frame (ROR-1rIg). Fused cDNA were
transfected into CHO cells and secreted recombinant protein was
immunoabsorbed using protein A sepharose. Absorbed protein was
immunoblotted with goat anti-ROR-1 antibody (R&D) or goat
anti-rabbit Ig antibody. KSHV K8.1 protein fused with rabbit Fc
region was also used for control. The purified recombinant ROR-1
was visualized with GelCode blue stain reagent (Pierce) staining
after SDS-PAGE.
[0038] FIG. 6B is a series of line and scatter plots showing
antibody reaction to ROR-1 detected by ELISA. Diluted sera were
reacted with coated ROR-1rIg and bound antibody was detected by
goat anti-human Ig antibody conjugated with HRP.
[0039] FIG. 6C is a series of line and scatter plots showing
antibody reaction to rabbit IgG detected by ELISA. Diluted sera
were reacted with coated rabbit IgG and bound antibody was detected
by goat anti-human Ig antibody conjugated with HRP.
[0040] FIG. 7 shows ROR-1 and Wnt5a activated NF-.kappa.B reporter
expression.
[0041] FIG. 7A is a series of bar graphs showing the effect of
ROR-1 on LEF/TCF1, NF-AT, and AP-1 activity. HEK293 cells were
transfected with indicated reporter construct and
.beta.-galactosidase vector along with expression vector of ROR-1
and Wnt5a.
[0042] FIG. 7B is a series of bar graphs showing the effect of
ROR-1 on NF-KB activity. HEK293 cells were transfected with
NF-.kappa.B reporter construct and .beta.-galactosidase vector
along with expression vector of ROR, Wnt5a, Wnt3, Wnt5b and
Wnt16.
[0043] FIG. 7C is a series of gel images showing in vitro binding
of ROR-1 and Wnt5a. Conditioned medium of transfectant with Wnt5a
tagged with HA was incubated with ROR-1rIg or rabbit IgG.
Immunoprecipitation and immunoblotting were done with indicated
materials.
[0044] FIG. 8 is a series of histograms showing gated CLL patients
and CD19+ and CD19+CD5+ cells.
[0045] FIG. 9 is a series of histograms showing gated normal
patients and CD19+ and CD19+CD5+ cells.
[0046] FIG. 10 is a series of histograms showing gated
"exceptional" normal patients and CD19+ and CD19+CD5+ cells.
[0047] FIG. 11 is a series of histograms showing gated CLL patients
and CD19+ and CD19+CD5+ cells.
[0048] FIG. 12 depicts the expression of 4A5 versus normals versus
CLLs and the gating effect.
[0049] FIG. 13 is a series of histograms showing different levels
of 4A5 expression on titrated CLL cells.
[0050] FIG. 14 is a series of histograms showing different levels
of 4A5 expression and that such cells can be purified using
magnetic beads and methods provided herein.
[0051] FIG. 15 is a series of histograms showing 4A5 expression on
various cancer cell lines.
[0052] FIG. 16A is an immunoblot of various human adult tissues for
expression of ROR-1, .beta.-actin or GAPDH, where the latter two
are used to control for protein loading.
[0053] FIG. 16B is an immunoblot of various human tissue for ROR-1
or .beta.-actin.
[0054] FIG. 16C are immunoprecipitation graphs based on the A5A
anti-ROR-1 mAb or an isotype control mAb to precipitate ROR-1
protein.
[0055] FIG. 16D are immunoprecipitation graphs based on the A5A
anti-ROR-1 mAB or an isotype control mAb to precipitate ROR-1
protein.
[0056] FIG. 17 are histograms showing ROR-1 expression in EW36, a
cell line that naturally expresses ROR1 (positive control; middle),
non-transfected P815 cells (right), and P815 cells transfected with
pcDNA3 ROR1. Cells were stained with Alexa-conjugated mouse IgG2b
(darker colored histogram) or Alexa-conjugated anti-ROR1 antibody
(light colored histogram) and analyzed by flow cytometry.
DETAILED DESCRIPTION OF THE INVENTION
[0057] As noted above, the instant invention provides new and
useful antibodies directed against ROR-1 protein. Full length
ROR-1, a surface receptor tyrosine kinase, is found in samples of
subjects with CLL, but not in blood or splenic lymphocytes of
nonleukemic patients or normal adults. The invention also provides
diagnostic and therapeutic antibodies, including monoclonal
antibodies, and related compositions and methods for use in the
diagnosis, management and treatment of disease. The ROR-1 antibody
described herein is more sensitive and more specific to ROR-1
expressing cancer cells than using a combination of several cell
surface markers that cannot exclude a small fraction of normal
cells.
[0058] Applicants have discovered expression of full-length ROR-1
in numerous cancer cell lines and samples, but not other tissues,
including blood or splenic lymphocytes of non-leukemic patients or
normal adult donors, and also generated mouse anti-sera against
full-length human ROR-1. Fukuda et al., Blood: ASH Annual Meeting
Abstracts 2004 104, Abstract 772 (2004) (incorporated herein by
reference in its entirety). The polypeptide and coding sequences
for ROR-1 have been reported elsewhere and are also incorporated
herein by this reference (see, e.g., Accession Nos.
NP.sub.--005003.1 and NM.sub.--005012.1).
[0059] ROR-1 Antibody
[0060] Certain embodiments comprise immunopeptides directed against
ROR-1 protein. The immunoglobulin peptides, or antibodies,
described herein are shown to bind to the ROR-1 protein. The ROR-1
binding activity is specific; the observed binding of antibody to
ROR-1 is not substantially blocked by non-specific reagents. These
ROR-1 specific antibodies can be used to differentiate between
ROR-1 cells and normal cells. The ROR-1 specific antibodies can
also be used in immunotherapy against a ROR-1 cancer and to
determine the response after therapy for a ROR-1 cancer.
[0061] Such immunopeptides can be raised in a variety of means
known to the art. For example, and as shown in the examples,
Ad-CD154 therapy induces humoral immunity against CLL, thus
allowing the derivation of immunoglobulin peptides specific against
ROR-1. The inventors have discovered that tandem injections of
Ad-CD154 induces antibody production against a novel cell surface
TAA of CLL B cells, orphan tyrosine kinase receptor ROR-1.
[0062] As used herein, the term antibody encompasses all types of
antibodies, e.g., polyclonal, monoclonal, and those produced by the
phage display methodology. Particularly preferred antibodies of the
invention are antibodies which have a relatively high degree of
affinity for ROR-1. In certain embodiments, the antibodies exhibit
an affinity for ROR-1 of about Kd<10.sup.-8 M.
[0063] Substantially purified generally refers to a composition
which is essentially free of other cellular components with which
the antibodies are associated in a non-purified, e.g., native state
or environment. Purified antibody is generally in a homogeneous
state, although it can be in either in a dry state or in an aqueous
solution. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography.
[0064] Substantially purified ROR-1-specific antibody will usually
comprise more than 80% of all macromolecular species present in a
preparation prior to admixture or formulation of the antibody with
a pharmaceutical carrier, excipient, adjuvant, buffer, absorption
enhancing agent, stabilizer, preservative, adjuvant or other
co-ingredient. More typically, the antibody is purified to
represent greater than 90% of all proteins present in a purified
preparation. In specific embodiments, the antibody is purified to
greater than 95% purity or may be essentially homogeneous wherein
other macromolecular species are not detectable by conventional
techniques.
[0065] Immunoglobulin peptides include, for example, polyclonal
antibodies, monoclonal antibodies, and antibody fragments. The
following describes generation of immunoglobulin peptides,
specifically ROR-1 antibodies, via methods that can be used by
those skilled in the art to make other suitable immunoglobulin
peptides having similar affinity and specificity which are
functionally equivalent to those used in the examples.
[0066] Polyclonal Antibodies
[0067] Polyclonal antibodies may be readily generated by one of
ordinary skill in the art from a variety of warm-blooded animals
such as horses, cows, various fowl, rabbits, mice, or rats.
Briefly, ROR-1 antigen is utilized to immunize the animal through
intraperitoneal, intramuscular, intraocular, or subcutaneous
injections, with an adjuvant such as Freund's complete or
incomplete adjuvant. Following several booster immunizations,
samples of serum are collected and tested for reactivity to ROR-1.
Particularly preferred polyclonal antisera will give a signal on
one of these assays that is at least three times greater than
background. Once the titer of the animal has reached a plateau in
terms of its reactivity to ROR-1, larger quantities of antisera may
be readily obtained either by weekly bleedings, or by
exsanguinating the animal.
[0068] Monoclonal Antibodies
[0069] Monoclonal antibody (mAb) technology can be used to obtain
mAbs to ROR-1. Briefly, hybridomas are produced using spleen cells
from mice immunized with ROR-1 antigens. The spleen cells of each
immunized mouse are fused with mouse myeloma Sp 2/0 cells, for
example using the polyethylene glycol fusion method of Galfre, G.
and Milstein, C., Methods Enzymol., 73:3-46 (1981). Growth of
hybridomas, selection in HAT medium, cloning and screening of
clones against antigens are carried out using standard methodology
(Galfre, G. and Milstein, C., Methods Enzymol., 73:3-46
(1981)).
[0070] HAT-selected clones are injected into mice to produce large
quantities of mAb in ascites as described by Galfre, G. and
Milstein, C., Methods Enzymol., 73:3-46 (1981), which can be
purified using protein A column chromatography (BioRad, Hercules,
Calif.). mAbs are selected on the basis of their (a) specificity
for ROR-1, (b) high binding affinity, (c) isotype, and (d)
stability.
[0071] mAbs can be screened or tested for ROR-1 specificity using
any of a variety of standard techniques, including Western Blotting
(Koren, E. et al., Biochim. Biophys. Acta 876:91-100 (1986)) and
enzyme-linked immunosorbent assay (ELISA) (Koren, E. et al.,
Biochim. Biophys. Acta 876:91-100 (1986)).
[0072] Humanized Antibodies
[0073] Humanized forms of mouse antibodies can be generated by
linking the CDR regions of non-human antibodies to human constant
regions by recombinant DNA techniques (see, e.g., Queen et al.,
Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989 and WO 90/07861,
each incorporated by reference). Human antibodies can be obtained
using phage-display methods (see, e.g., Dower et al., WO 91/17271;
McCafferty et al., WO 92/01047). In these methods, libraries of
phage are produced in which members display different antibodies on
their outersurfaces. Antibodies are usually displayed as Fv or Fab
fragments. Phage displaying antibodies with a desired specificity
may be selected by affinity enrichment.
[0074] Human antibodies may be selected by competitive binding
experiments, or otherwise, to have the same epitope specificity as
a particular mouse antibody. Using these techniques, a humanized
ROR-1 antibody having the human IgG1 constant region domain and the
human kappa light chain constant region domain with the mouse heavy
and light chain variable regions. The humanized antibody has the
binding specificity of a mouse ROR-1 mAb, specifically the 45A mAb
described in Example 9.
[0075] Antibody Fragments
[0076] It may be desirable to produce and use functional fragments
of a mAb for a particular application. The well-known basic
structure of a typical IgG molecule is a symmetrical tetrameric
Y-shaped molecule of approximately 150,000 to 200,000 daltons
consisting of two identical light polypeptide chains (containing
about 220 amino acids) and two identical heavy polypeptide chains
(containing about 440 amino acids). Heavy chains are linked to one
another through at least one disulfide bond. Each light chain is
linked to a contiguous heavy chain by a disulfide linkage. An
antigen-binding site or domain is located in each arm of the
Y-shaped antibody molecule and is formed between the amino terminal
regions of each pair of disulfide linked light and heavy chains.
These amino terminal regions of the light and heavy chains consist
of approximately their first 110 amino terminal amino acids and are
known as the variable regions of the light and heavy chains. In
addition, within the variable regions of the light and heavy chains
there are hypervariable regions which contain stretches of amino
acid sequences, known as complementarity determining regions
(CDRs). CDRs are responsible for the antibody's specificity for one
particular site on an antigen molecule called an epitope. Thus, the
typical IgG molecule is divalent in that it can bind two antigen
molecules because each antigen-binding site is able to bind the
specific epitope of each antigen molecule. The carboxy terminal
regions of light and heavy chains are similar or identical to those
of other antibody molecules and are called constant regions. The
amino acid sequence of the constant region of the heavy chains of a
particular antibody defines what class of antibody it is, for
example, IgG, IgD, IgE, IgA or IgM. Some classes of antibodies
contain two or more identical antibodies associated with each other
in multivalent antigen-binding arrangements.
[0077] Fab and F(ab').sub.2 fragments of mAbs that bind ROR-1 can
be used in place of whole mAbs. Because Fab and F(ab').sub.2
fragments are smaller than intact antibody molecules, more
antigen-binding domains are available than when whole antibody
molecules are used. Proteolytic cleavage of a typical IgG molecule
with papain is known to produce two separate antigen binding
fragments called Fab fragments which contain an intact light chain
linked to an amino terminal portion of the contiguous heavy chain
via by disulfide linkage. The remaining portion of the
papain-digested immunoglobin molecule is known as the Fc fragment
and consists of the carboxy terminal portions of the antibody left
intact and linked together via disulfide bonds. If an antibody is
digested with pepsin, a fragment known as an F(ab').sub.2 fragment
is produced which lacks the Fc region but contains both
antigen-binding domains held together by disulfide bonds between
contiguous light and heavy chains (as Fab fragments) and also
disulfide linkages between the remaining portions of the contiguous
heavy chains (Handbook of Experimental Immunology. Vol 1:
Immunochemistry, Weir, D. M., Editor, Blackwell Scientific
Publications, Oxford (1986)).
[0078] Recombinant DNA methods have been developed which permit the
production and selection of recombinant immunoglobulin peptides
which are single chain antigen-binding polypeptides known as single
chain Fv fragments (ScFvs or ScFv antibodies). Further, ScFvs can
be dimerized to produce a diabody. ScFvs bind a specific epitope of
interest and can be produced using any of a variety of recombinant
bacterial phage-based methods, for example as described in Lowman
et al. (1991) Biochemistry, 30, 10832-10838; Clackson et al. (1991)
Nature 352, 624-628; and Cwirla et al. (1990) Proc. Natl. Acad.
Sci. USA 87, 6378-6382. These methods are usually based on
producing genetically altered filamentous phage, such as
recombinant M13 or fd phages, which display on the surface of the
phage particle a recombinant fusion protein containing the
antigen-binding ScFv antibody as the amino terminal region of the
fusion protein and the minor phage coat protein g3p as the carboxy
terminal region of the fusion protein. Such recombinant phages can
be readily grown and isolated using well-known phage methods.
Furthermore, the intact phage particles can usually be screened
directly for the presence (display) of an antigen-binding ScFv on
their surface without the necessity of isolating the ScFv away from
the phage particle.
[0079] To produce an ScFv, standard reverse transcriptase protocols
are used to first produce cDNA from mRNA isolated from a hybridoma
that produces an mAb for ROR-1 antigen. The cDNA molecules encoding
the variable regions of the heavy and light chains of the mAb can
then be amplified by standard polymerase chain reaction (PCR)
methodology using a set of primers for mouse immunoglobulin heavy
and light variable regions (Clackson (1991) Nature, 352, 624-628).
The amplified cDNAs encoding mAb heavy and light chain variable
regions are then linked together with a linker oligonucleotide in
order to generate a recombinant ScFv DNA molecule. The ScFv DNA is
ligated into a filamentous phage plasmid designed to fuse the
amplified cDNA sequences into the 5' region of the phage gene
encoding the minor coat protein called g3p. Escherichia coli
bacterial cells are than transformed with the recombinant phage
plasmids, and filamentous phage grown and harvested. The desired
recombinant phages display antigen-binding domains fused to the
amino terminal region of the minor coat protein. Such "display
phages" can then be passed over immobilized antigen, for example,
using the method known as "panning", see Parmley and Smith (1989)
Adv. Exp. Med. Biol. 251, 215-218; Cwirla et al. (1990) Proc. Natl.
Acad. Sci. USA 87, 6378-6382, to adsorb those phage particles
containing ScFv antibody proteins that are capable of binding
antigen. The antigen-binding phage particles can then be amplified
by standard phage infection methods, and the amplified recombinant
phage population again selected for antigen-binding ability. Such
successive rounds of selection for antigen-binding ability,
followed by amplification, select for enhanced antigen-binding
ability in the ScFvs displayed on recombinant phages. Selection for
increased antigen-binding ability may be made by adjusting the
conditions under which binding takes place to require a tighter
binding activity. Another method to select for enhanced
antigen-binding activity is to alter nucleotide sequences within
the cDNA encoding the binding domain of the ScFv and subject
recombinant phage populations to successive rounds of selection for
antigen-binding activity and amplification (see Lowman et al.
(1991) Biochemistry 30, 10832-10838; and Cwirla et al. (1990) Proc.
Natl. Acad. Sci. USA 87, 6378-6382).
[0080] Once an ScFv is selected, the recombinant ROR-1 antibody can
be produced in a free form using an appropriate vector in
conjunction with E. coli strain HB2151. These bacteria actually
secrete ScFv in a soluble form, free of phage components
(Hoogenboom et al. (1991) Nucl. Acids Res. 19, 4133-4137). The
purification of soluble ScFv from the HB2151 bacteria culture
medium can be accomplished by affinity chromatography using antigen
molecules immobilized on a solid support such as AFFIGEL.TM.
(BioRad, Hercules, Calif.).
[0081] Other developments in the recombinant antibody technology
demonstrate possibilities for further improvements such as
increased avidity of binding by polymerization of ScFvs into dimers
and tetramers (see Holliger et al. (1993) Proc. Natl. Acad. Sci.
USA 90, 6444-6448).
[0082] Because ScFvs are even smaller molecules than Fab or
F(ab').sub.2 fragments, they can be used to attain even higher
densities of antigen binding sites per unit of surface area when
immobilized on a solid support material than possible using whole
antibodies, F(ab').sub.2, or Fab fragments. Furthermore,
recombinant antibody technology offers a more stable genetic source
of antibodies, as compared with hybridomas. Recombinant antibodies
can also be produced more quickly and economically using standard
bacterial phage production methods.
[0083] Recombinant Antibody Production
[0084] To produce antibodies described herein recombinantly,
nucleic acids encoding light and heavy chain variable regions,
optionally linked to constant regions, are inserted into expression
vectors. The light and heavy chains can be cloned in the same or
different expression vectors. For example, the heavy and light
chains of SEQ ID NOs: 1-5 can be used according to the present
invention. The teachings of U.S. Pat. No. 6,287,569 to Kipps et
al., incorporated herein by reference in its entirety, and the
methods provided herein can readily be adapted by those of skill in
the art to create the vaccines of the present invention. The DNA
segments encoding antibody chains are operably linked to control
sequences in the expression vector(s) that ensure the expression of
antibody chains. Such control sequences include a signal sequence,
a promoter, an enhancer, and a transcription termination sequence.
In one embodiment, the
[0085] Expression vectors are typically replicable in the host
organisms either as episomes or as an integral part of the host
chromosome. E. coli is one procaryotic host particularly useful for
expressing antibodies of the present invention. Other microbial
hosts suitable for use include bacilli, such as Bacillus subtilus,
and other enterobacteriaceae, such as Salmonella, Serratia, and
various Pseudomonas species. In these prokaryotic hosts, one can
also make expression vectors, which typically contain expression
control sequences compatible with the host cell (e.g., an origin of
replication) and regulatory sequences such as a lactose promoter
system, a tryptophan (trp) promoter system, a beta-lactamase
promoter system, or a promoter system from phage lambda. Other
microbes, such as yeast, may also be used for expression.
Saccharomyces is a preferred host, with suitable vectors having
expression control sequences, such as promoters, including
3-phosphoglycerate kinase or other glycolytic enzymes, and an
origin of replication, termination sequences and the like as
desired. Mammalian tissue cell culture can also be used to express
and produce the antibodies of the present invention (see, e.g.,
Winnacker, From Genes to Clones VCH Publishers, N.Y., 1987).
Eukaryotic cells are preferred, because a number of suitable host
cell lines capable of secreting intact antibodies have been
developed. Preferred suitable host cells for expressing nucleic
acids encoding the immunoglobulins of the invention include: monkey
kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line; baby hamster kidney cells (BHK, ATCC CCL
10); Chinese hamster ovary-cells (CHO); mouse sertoli cells; monkey
kidney cells (CV1 ATCC CCL 70); african green monkey kidney cells
(VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA,
ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat
liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC
CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor
(MMT 060562, ATCC CCL51); and TRI cells.
[0086] The vectors containing the polynucleotide sequences of
interest (e.g., the heavy and light chain encoding sequences and
expression control sequences) can be transferred into the host
cell. Calcium chloride transfection is commonly utilized for
prokaryotic cells, whereas calcium phosphate treatment or
electroporation can be used for other cellular hosts (see, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Press, 2nd ed., 1989). When heavy and light chains
are cloned on separate expression vectors, the vectors are
co-transfected to obtain expression and assembly of intact
immunoglobulins. After introduction of recombinant DNA, cell lines
expressing immunoglobulin products are cell selected. Cell lines
capable of stable expression are preferred (i.e., undiminished
levels of expression after fifty passages of the cell line).
[0087] Once expressed, the whole antibodies, their dimers,
individual light and heavy chains, or other immunoglobulin forms of
the present invention can be purified according to standard
procedures of the art, including ammonium sulfate precipitation,
affinity columns, column chromatography, gel electrophoresis and
the like (see, e.g., Scopes, Protein Purification, Springer-Verlag,
N.Y., 1982). Substantially pure immunoglobulins of at least about
90 to 95% homogeneity are preferred, and 98 to 99% or more
homogeneity most preferred.
[0088] Labeled Antibody
[0089] A labeled antibody or a detectably labeled antibody is
generally an antibody (or antibody fragment which retains binding
specificity), having an attached detectable label. The detectable
label is normally attached by chemical conjugation, but where the
label is a polypeptide, it could alternatively be attached by
genetic engineering techniques. Methods for production of
detectably labeled proteins are well known in the art. Detectable
labels known in the art include radioisotopes, fluorophores,
paramagnetic labels, enzymes (e.g., horseradish peroxidase), or
other moieties or compounds which either emit a detectable signal
(e.g., radioactivity, fluorescence, color) or emit a detectable
signal after exposure of the label to its substrate. Various
detectable label/substrate pairs (e.g., horseradish
peroxidase/diaminobenzidine, avidin/streptavidin,
luciferase/luciferin), methods for labeling antibodies, and methods
for using labeled antibodies are well known in the art (see, for
example, Harlow and Lane, eds., 1988, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.) Another technique which may also result in greater
sensitivity consists of coupling the antibodies to low molecular
weight haptens. These haptens can then be specifically detected by
means of a second reaction. For example, it is common to use such
haptens as biotin, which reacts with avidin, or dinitrophenyl,
pyridoxal, and fluorescein, which can react with specific
antihapten antibodies.
[0090] Diagnosis of ROR-1 Cancer
[0091] The ROR-1 antibodies described herein can be used to
differentiate between ROR-1 expressing cells and normal cells and,
thus, can be used to detect and/or diagnose disease in subjects.
ROR-1 expressing cancer cells include CLL and other lymphoma (e.g.
Burkitt's), renal cell carcinoma, colon adenocarcinoma, colorectal
(see, e.g., FIG. 15).
[0092] The methods for detecting such disease generally include
contacting a sample from a subject having, or at risk of having, a
lymphoma with a reagent that detects ROR-1, and detecting the
reaction of the reagent. Within these methods, detection of a
reaction is indicative of the presence and/or quantity of ROR-1 in
the sample. The reaction of the reagent with the sample is then
compared to a control. Any biological sample which may contain a
detectable amount of ROR-1 can be used. Examples of biological
samples of use with the invention are blood, serum, plasma, urine,
mucous, feces, cerebrospinal fluid, pleural fluid, ascites, and
sputum samples. Tissue or cell samples can also be used with the
subject invention. These samples can be obtained by many methods
such as cellular aspiration, or by surgical removal of a biopsy
sample. The level of ROR-1 in the sample can be compared with the
level in a sample not affected by the targeted disorder or
condition. Control samples not affected by a targeted disease
processes can be taken from the same subject, or can be from a
normal control subject not affected by the disease process, or can
be from a cell line.
[0093] Contacting the sample and anti-ROR-1 antibody generally
includes incubation under conditions which allow contact in
solution and/or solid phase between the reagent and sample.
Detection can be performed by any means suitable to identify the
interaction of the reagent with ROR-1. In one embodiment, when the
reagent is an antibody, the antibody can be detectably labeled.
Detectable labels are well known in the art, and include
radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g.,
horseradish peroxidase), or other moieties or compounds which
either emit a detectable signal (e.g., radioactivity, fluorescence,
color) or emit a detectable signal after exposure of the label to
its substrate. Alternatively, when the reagent is an antibody,
detection can be performed using a second antibody which is
detectably labeled which recognizes the antibody that binds ROR-1.
The antibody may also be biotinylated, and a second avidinated
label used to determine the presence of the biotinylated reagent
which detects ROR-1.
[0094] The antibodies of the invention are suited for use, for
example, in immunoassays in which they can be utilized in liquid
phase or bound to a solid phase carrier. The antibodies employed in
these immunoassays can be detectably labeled in various ways.
Examples of types of immunoassays which can effectively employ
antibodies of the invention are, competitive and non-competitive
immunoassays, in either a direct or indirect format. Examples of
such immunoassays include a radioimmunoassay (RIA), and a sandwich
(immunometric) assay. Those of skill in the art will readily
discern additional immunoassay formats useful within the
invention.
[0095] Other immunoassays for use within the invention include
"forward" assays for the detection of a protein in which a first
anti-protein antibody (e.g., an anti-ROR-1 antibody) bound to a
solid phase support is contacted with the test sample. After a
suitable incubation period, the solid phase support is washed to
remove unbound protein. A second, distinct anti-protein antibody is
then added which is specific for a portion of the specific protein
not recognized by the first antibody. The second antibody is
preferably detectable. After a second incubation period to permit
the detectable antibody to complex with the specific protein bound
to the solid phase support through the first antibody, the solid
phase support is washed a second time to remove the unbound
detectable antibody. Alternatively, the second antibody may not be
detectable. In this case, a third detectable antibody, which binds
the second antibody is added to the system. This type of "forward
sandwich" assay may be a simple yes/no assay to determine whether
binding has occurred or may be made quantitative by comparing the
amount of detectable antibody with that obtained in a control.
[0096] Other types of immunometric assays are the so-called
"simultaneous" and "reverse" assays. A simultaneous assay involves
a single incubation step wherein the first antibody bound to the
solid phase support, the second, detectable antibody and the test
sample are added at the same time. After the incubation is
completed, the solid phase support is washed to remove unbound
proteins. The presence of detectable antibody associated with the
solid support is then determined as it would be in a conventional
"forward sandwich" assay. The simultaneous assay may also be
adapted in a similar manner for the detection of antibodies in a
test sample. The "reverse" assay comprises the stepwise addition of
a solution of detectable antibody to the test sample followed by an
incubation period and the addition of antibody bound to a solid
phase support after an additional incubation period. The solid
phase support is washed in conventional fashion to remove unbound
protein/antibody complexes and unreacted detectable antibody. The
determination of detectable antibody associated with the solid
phase support is then determined as in the "simultaneous" and
"forward" assays. The reverse assay may also be adapted in a
similar manner for the detection of antibodies in a test
sample.
[0097] The antibody component of immunometric assays described
herein may be added to a solid phase support capable of
immobilizing proteins. By "solid phase support" or "support" is
intended any material capable of binding proteins. Well-known solid
phase supports include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses (including nitrocellulose sheets and filters),
polyacrylamides, agaroses, and magnetite. The nature of the support
can be either soluble to some extent or insoluble for the purposes
of the present invention. The support configuration may be
spherical, as in a bead, or cylindrical, as in the inside surface
of a test tube or the external surface of a rod. Alternatively, the
surface may be flat such as a sheet, test strip, etc. Those skilled
in the art will know many other suitable "solid phase supports" for
binding proteins or will be able to ascertain the same by use of
routine experimentation. A preferred solid phase support is a
96-well microtiter plate. For immunoassay and immunodiagnostic
purposes, the antibodies of the invention can be bound to many
different carriers, both soluble and insoluble, and can be used to
detect the presence of an antigen comprising ROR-1 (or fragments,
derivatives, conjugates, homologues, or variants thereof). Those
skilled in the art will discern other suitable carriers for binding
antibodies useful within the invention. In addition, there are many
different labels and methods of labeling known to those of ordinary
skill in the art. Examples of the types of labels which can be used
in the present invention include enzymes, radioisotopes,
fluorescent compounds, colloidal metals, chemiluminescent
compounds, phosphorescent compounds, and bioluminescent compounds,
as described above.
[0098] In using the antibodies described herein for the in vitro or
in vivo detection of ROR-1, the detectably labeled antibody is
provided in an amount which is diagnostically effective. Thus, an
amount of detectably labeled antibody is contacted or administered
in sufficient quantity to enable detection of ROR-1 in the subject
sample to be assayed.
[0099] Within more detailed diagnostic methods of the invention, in
vivo immunodiagnostic tools are provided, as exemplified by
immunoscintigraphic methods and compositions. Immunoscintigraphy
(IS) is discussed in detail in P. Lechner et al., Dis Colon Rectum
1993; 36:930-935 and F. L. Moffet et al., J Clin Oncol 14:2295-2305
(1966). IS (or radioscintigraphy) employs radioactive-labeled
antibody, typically Fab' fragments (Goldenberg et al.; Eur J Nucl
Med 1989; 15:426), to recognize defined epitopes of targeted
proteins. Fab' fragments of the antibodies provided herein,
comprising immunoglobulins of the IgGI fraction that have their Fc
portions removed, are highly capable of targeting epitopes on
proCPR, activated CPR, and/or inactivated CPR in a test sample or
subject. Because these Fab' fragments have minimal antigenity, they
cause neither human antimouse antibody response, nor any allergic
reactions of unpredictable nature. The smaller molecular weight of
Fab' fragments compared with intact antibody allows the fragment to
leave the intravascular space and target a broader array of in vivo
compartments for diagnostic purposes.
[0100] For radioscintigraphy, an anti-ROR-1 radioactive monoclonal
antibody is typically injected into a patient for identifying,
measuring, and/or localizing ROR-1 in the subject, (see, e.g.,
Delaloye et al., Seminars in Nuclear Medicine 25(2):144-164, 1995).
In radioimaging with monoclonal antibodies, a chemically modified
(chelate) form of the monoclonal antibody is typically prepared and
stored as a relatively stable product. To be used clinically,
however, the monoclonal antibody sample must be mixed with a
radioactive metal, such as .sup.99Tc, then purified to remove
excess, unbound radioactive metal, and then administered to a
patient within 6 hours, (see, e.g., Eckelman et al., Nuc. Med.
Biol. 16: 171-176, 1989). Radioisotopes, for example .sup.99Tc, an
isotope with a short physical half-life and high photon abundance,
can be administered at high doses and allow early imaging with a
gamma camera. This is very suitable for use in conjunction with
Fab' fragments, the half-lives of which are also short.
[0101] Monitoring of a ROR-1 Cancer and Cancer Therapy
[0102] Further, the anti-ROR-1 antibodies described herein can be
used in vitro and in vivo to monitor the appearance, status,
course, or treatment of a ror-1 cancer in a subject. For example,
by measuring an increase or decrease in the amount of ROR-1 in a
subject (optionally in comparison to control levels in a normal
subject or sample), the appearance, status, course, or treatment of
the cancer or condition in the subject number can be observed or
evaluated. Based on these and comparable diagnostic methods, it is
further possible to determine whether a particular therapeutic
regimen, such as a treatment regimen employing antibodies of the
invention directed against the cancer is effective. Methods of
detecting and/or quantifying levels of ROR-1 and corresponding
cancer disease state are as described above.
[0103] Therapeutic Treatment of Lymphoma
[0104] ROR-1 agonists can be employed as therapeutic or
prophylactic pharmacological agents in any subject in which it is
desirable to administer, in vitro, ex vivo, or in vivo the subject
agonists that bind ROR-1. Typical subjects for treatment or
management according to the methods herein are subjects presenting
with a ROR-1 cancer. The agonists described herein specifically
recognize ROR-1 protein, found in lymphoma samples but not
expressed in cells of normal adults, and therefore can be used for
detecting and/or neutralizing these biomolecules, and/or blocking
their interactions with other biomolecules, in vitro or in vivo.
Examples of such ROR-1 agonists include antibodies, small molecule
inhibitors, antisense RNA, and siRNA.
[0105] While under no obligation to provide a mechanism of action,
it is thought that ROR-1 can serve as a receptor for Wnt5a to
trigger the NF-kappa B pathway, which pathway is implicated in
oncogenesis. See e.g. Example 12. Thus, the ROR-1 gene, which plays
a role in disease pathogenesis and/or progression, encodes a
protein that can be targeted by immune therapy for patients with a
ROR-1 cancer.
[0106] Antibodies
[0107] In certain therapeutic embodiments, the selected antibody
will typically be an anti-ROR-1 antibody, which may be administered
alone, or in combination with, or conjugated to, one or more
combinatorial therapeutic agents. When the antibodies described
herein are administered alone as therapeutic agents, they may exert
a beneficial effect in the subject by a variety of mechanisms. In
certain embodiments, monoclonal antibodies that specifically bind
ROR-1 are purified and administered to a patient to neutralize one
or more forms of ROR-1, to block one or more activities of ROR-1,
or to block or inhibit an interaction of one or more forms of ROR-1
with another biomolecule.
[0108] The immunotherapeutic reagents of the invention may include
humanized antibodies, and can be combined for therapeutic use with
additional active or inert ingredients, e.g., in conventional
pharmaceutically acceptable carriers or diluents, e.g., immunogenic
adjuvants, and optionally with adjunctive or combinatorially active
agents such as anti-inflammatory ant anti-fibrinolytic drugs.
[0109] In other embodiments, therapeutic antibodies described
herein are coordinately administered with, co-formulated with, or
coupled to (e.g., covalently bonded) a combinatorial therapeutic
agent, for example a radionuclide, a differentiation inducer, a
drug, or a toxin. Various known radionuclides can be employed,
including .sup.90Y, .sup.123I, .sup.125I, .sup.131I, .sup.186Re,
.sup.188Re, and .sup.211At. Useful drugs for use in such
combinatorial treatment formulations and methods include
methotrexate, and pyrimidine and purine analogs. Suitable
differentiation inducers include phorbol esters and butyric acid.
Suitable toxins include ricin, abrin, diptheria toxin, cholera
toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed
antiviral protein. These combinatorial therapeutic agents can be
coupled to an anti-ROR-1 antibody either directly or indirectly
(e.g., via a linker group). A direct reaction between an agent and
an antibody is possible when each possesses a substituent capable
of reacting with the other. For example, a nucleophilic group, such
as an amino or sulfhydryl group, on one may be capable of reacting
with a carbonyl-containing group, such as an anhydride or an acid
halide, or with an alkyl group containing a good leaving group
(e.g., a halide) on the other. Alternatively, it may be desirable
to couple a combinatorial therapeutic agent and an antibody via a
linker group as a spacer to distance an antibody from the
combinatorial therapeutic agent in order to avoid interference with
binding capabilities. A linker group can also serve to increase the
chemical reactivity of a substituent on an agent or an antibody,
and thus increase the coupling efficiency. It will be further
evident to those skilled in the art that a variety of bifunctional
or polyfunctional reagents, both homo- and hetero-functional (such
as those described in the catalog of the Pierce Chemical Co.,
Rockford, Ill.), may be employed as a linker group. Coupling may be
affected, for example, through amino groups, carboxyl groups,
sulfhydryl groups or oxidized carbohydrate residues.
[0110] Where a therapeutic agent is more potent when free from the
antibody portion of the immunoconjugates described herein, it may
be desirable to use a linker group which is cleavable during or
upon internalization into a cell. A number of different cleavable
linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include
cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.
4,489,710, to Spitler), by irradiation of a photolabile bond (e.g.,
U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045,
to Kohn et al.), by serum complement-mediated hydrolysis (e.g.,
U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed
hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.) It
may also be desirable to couple more than one agent to an
anti-ROR-1 antibody. In one embodiment, multiple molecules of an
agent are coupled to one antibody molecule. In another embodiment,
more than one type of agent may be coupled to one antibody.
Regardless of the particular embodiment, immunoconjugates with more
than one agent may be prepared in a variety of ways. For example,
more than one agent may be coupled directly to an antibody
molecule, or linkers which provide multiple sites for attachment
can be used. Alternatively, a carrier can be used.
[0111] A variety of routes of administration for the antibodies and
immunoconjugates may be used. Typically, administration is
intravenous, intramuscular, or subcutaneous.
[0112] It will be evident that the precise dose of the
antibody/immunoconjugate will vary depending upon such factors as
the antibody used, the antigen density, and the rate of clearance
of the antibody. A safe and effective amount of an anti-ROR-1 agent
is, for example, that amount that would cause the desired
therapeutic effect in a patient while minimizing undesired side
effects. Generally, a therapeutically effective amount is that
sufficient to promote production of one or more cytokines and/or to
cause complement-mediated or antibody-dependent cellular
cytotoxicity. The dosage regimen will be determined by skilled
clinicians, based on factors such as the exact nature of the
condition being treated, the severity of the condition, the age and
general physical condition of the patient, and so on.
[0113] siRNA
[0114] In certain therapeutic embodiments, the ROR-1 agonist is
siRNA. The levels of ROR-1 can be down-regulated by RNA
interference by administering to the patient a therapeutically
effective amount of small interfering RNAs (siRNA) specific for
ROR-1. siRNA specific for ROR-1 can be produced commercially from a
variety of sources, such as Ambion (Austin, Tex.). The siRNA can be
administered to the subject by any means suitable for delivering
the siRNA to the blood. For example, the siRNA can be administered
by gene gun, electroporation, or by other suitable parenteral or
enteral administration routes, such as intravitreous injection.
[0115] RNA interference is the process by which double stranded RNA
(dsRNA) specifically suppresses the expression of a gene bearing
its complementary sequence. Suppression of the ROR-1 gene inhibits
the production of the ROR-1 protein. Upon introduction, the long
dsRNAs enter a cellular pathway that is commonly referred to as the
RNA interference (RNAi) pathway. First, the dsRNAs get processed
into 20-25 nucleotide (nt) small interfering RNAs (siRNAs) by an
RNase III-like enzyme called Dicer (initiation step). Then, the
siRNAs assemble into endoribonuclease-containing complexes known as
RNA-induced silencing complexes (RISCs), unwinding in the process.
The siRNA strands subsequently guide the RISCs to complementary RNA
molecules, where they cleave and destroy the cognate RNA (effecter
step). Cleavage of cognate RNA takes place near the middle of the
region bound by the siRNA strand. Preferably, the siRNA comprises
short double-stranded RNA from about 17 nucleotides to about 29
nucleotides in length, preferably from about 19 to about 25
nucleotides in length, that are targeted to the target mRNA.
[0116] As an example, an effective amount of the siRNA can be an
amount sufficient to cause RNAi-mediated degradation of the target
ROR-1 mRNA, or an amount sufficient to inhibit the progression of a
lymphoma in a subject. One skilled in the art can readily determine
an effective amount of the siRNA of the invention to be
administered to a given subject by taking into account factors such
as the size and weight of the subject; the extent of the
neovascularization or disease penetration; the age, health and sex
of the subject; the route of administration; and whether the
administration is regional or systemic. Generally, an effective
amount of siRNA comprises an intercellular concentration of from
about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM
to about 50 nM, more preferably from about 2.5 nM to about 10 nM.
It is contemplated that greater or lesser amounts of siRNA can be
administered.
[0117] The siRNA can be targeted to any stretch of approximately
19-25 contiguous nucleotides in any of the ROR-1 mRNA target
sequences. Target sequences can be selected from, for example, the
sequence of ROR-1, Genebank accession number: NM.sub.--005012.
Searches of the human genome database (BLAST) can be carried out to
ensure that selected siRNA sequence will not target other gene
transcripts. Techniques for selecting target sequences for siRNA
are given, for example, in Elbashir et al. ((2001) Nature 411,
494-498). Thus, the sense strand of the present siRNA comprises a
nucleotide sequence identical to any contiguous stretch of about 19
to about 25 nucleotides in the target mRNA of ROR-1. Generally, a
target sequence on the target mRNA can be selected from a given
cDNA sequence corresponding to the target mRNA, preferably
beginning 50 to 100 nt downstream (i.e., in the 3' direction) from
the start codon. The target sequence can, however, be located in
the 5' or 3' untranslated regions, or in the region nearby the
start codon.
[0118] Antisense
[0119] In certain therapeutic embodiments, the ROR-1 agonist is an
antisense oligonucleotide. The levels of ROR-1 can be
down-regulated by administering to the patient a therapeutically
effective amount of an antisense oligonucleotide specific for ROR-1
mRNA. The antisense oligonucleotide specific for ROR-1 mRNA may
span the region adjacent to the initiation site of ROR-1
translation.
[0120] An effective amount of the antisense oligonucleotide
specific for ROR-1 mRNA as isolated in a purified form may is
generally that amount capable of inhibiting the production of ROR-1
or reducing the amount produced or the rate of production of ROR-1
such that a reduction in symptoms of lymphoma occurs. Antisense
oligonucleotides can be administered via intravitreous injection at
a concentration of about 10 .mu.g/day to about 3 mg/day. For
example, administered dosage can be about 30 .mu.g/day to about 300
.mu.g/day. As another example, ROR-1 antisense oligonucleotide can
be administered at about 100 .mu.g/day. Administration of antisense
oligonucleotides can occur as a single event or over a time course
of treatment. For example, ROR-1 antisense oligonucleotides can be
injected daily, weekly, bi-weekly, or monthly. Time course of
treatment can be from about a week to about a year or more. In one
example, ROR-1 antisense oligonucleotides are injected daily for
one month. In another example, antisense oligonucleotides are
injected weekly for about 10 weeks. In a further example, ROR-1
antisense oligonucleotides are injected every 6 weeks for 48
weeks.
[0121] Vaccines
[0122] As will be clear from the description herein of anti-ROR-1
antibody, the present invention also provides for use of ROR-1 in
vaccines against diseases, such as a lymphoma, e.g., CLL, that
involve the expression of ROR-1. Because normal adult tissues do
not appear to express ROR-1, it represents a tumor-specific antigen
that can be targeted in active immune therapy. For example, the
levels of ROR-1 can be down-regulated by administering to the
patient a therapeutically effective amount of a ROR-1
polynucleotide or polypeptide that produces in animals a protective
or therapeutic immune response against ROR-1 and the effects of its
expression. The vaccines can include polynucleotides or
polypeptides. Methods of using such polynucleotides and/or
polypeptides include use in vaccines and for generating antibodies
against the polypeptides, such as those expressed by the
polynucleotides. The polynucleotides can be a ROR-1 gene, or a
variant or fragment thereof. The polypeptides can be a ROR-1
protein, or a variant or fragment thereof. In certain aspects, the
ROR-1 polynucleotide fragment can be a fragment comprising a
fragment of the ROR-1 gene. Such polynucleotide fragments can be
comprised by a vector. A cell can be transformed and/or transfected
by such polynucleotides and vectors and in certain aspects, the
polynucleotides and vectors can express polypeptides of the
invention. Typically the vaccine composition includes a
pharmaceutically acceptable carrier or diluent. The teachings of
U.S. Pat. No. 6,287,569 to Kipps et al., incorporated herein by
reference in its entirety, can readily be adapted by those of skill
in the art to create the vaccines of the present invention.
[0123] When describing a vaccine, a "polynucleotide variant" refers
to any degenerate nucleotide sequence. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptide
encoded by the reference sequence. For example, a variant
polynucleotide consisting of 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99% to the
polynucleotide consisting of ibpA. A "polynucleotide fragment" of a
ROR-1 polynucleotide is a portion of a ROR-1 polynucleotide that is
less than full-length and comprises at least a minimum length
capable of hybridizing specifically with a native ROR-1
polynucleotide under stringent hybridization conditions. The length
of such a fragment is preferably at least 15 nucleotides, more
preferably at least 20 nucleotides, and most preferably at least 30
nucleotides of a native ibpA polynucleotide sequence. A
"polypeptide variant" refers to a polypeptide of differs in amino
acid sequence from the ibpA polypeptide. Generally, differences are
limited so that the sequences of the reference polypeptide and the
variant are closely similar overall and, in many regions,
identical. A variant polypeptide may differ in amino acid sequence
by one or more substitutions, additions, deletions in any
combination. A substituted or inserted amino acid residue may or
may not be one encoded by the genetic code. Finally, a "polypeptide
fragment" refers to any polypeptide of a portion of a ibpA
polypeptide that is less than full-length (e.g., a polypeptide
consisting of 5, 10, 15, 20, 30, 40, 50, 75, 100 or more amino
acids of a native ROR-1 protein), and preferably retains at least
one functional activity of a native ROR-1 protein.
[0124] DNA Vaccines for ROR-1
[0125] Polypeptides with Arg at their N-terminus have a shorter
half-life in the cytosol than those with a Met residue, provided
that the polypeptide has a lysine residue to function as an
ubiquitin acceptor site, spaced within 20 amino acids of the
N-terminus. Plasmids encoding antigens targeted for rapid
degradation by the proteasome are more effective than plasmids
encoding the native protein in inducing CTL responses against cells
expressing the target antigen.
[0126] Vectors have been constructed that encode a chimeric ROR1
protein with ubiquitin located at the amino terminus separated from
ROR1 by an intervening codon for Met, and one with a codon for the
destabilizing amino acid Arg and an in-frame insert of a segment of
lacI. This segment contains a lysine residue spaced optimally from
the N-terminus. Both constructs contain a sequence from the
ubiquitine gene (SEQ ID NO: 6), followed by methionine or arginine
sequence, followed by a LacI sequence (SEQ ID NO 7), and finally
followed by the ROR-1 cDNA sequence (SEQ ID NO: 8). As detailed
further in Example 16, the constructs are useful in ROR-1 DNA
vaccines, with the arginine construct being expected to cause rapid
degradation of the protein and thus a more predominant cellular
immune response.
[0127] Many embodiments of the invention are provided through well
known protocols established in the art. For example, the following
references provide multiple protocols which may be adapted for use
with anti-ROR-1 antibody: Vernon, S. K., Lawrence, W. C., Long, C.
A., Cohen, G. H., and Rubin, B. A. Herpesvirus vaccine development:
Studies of virus morphological components. In New Trends and
Developments in Vaccines, ed. by A. Voller and H. Friedman. Chapter
13, pp. 179-210. MTP Press, Ltd., Lancaster (1978); Sambrook et al.
(1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3,
Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.,
("Sambrook"); and Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(e.g., current through 1999, e.g., at least through supplement 37)
("Ausubel")), each of which are incorporated herein by reference in
its entirety. With respect to vaccine technologies, U.S. Patent
Application Nos. 20040253240 and 20030124141, are incorporated
herein by reference in their entirety. These references also
provide one of skill in the art instructions how to make and use
the polynucleotides and polypeptides of the present invention for
active and passive vaccines. Those of skill in the art will readily
recognize how to adapt the disclosures of these references to the
present polynucleotides and polypeptides of the present
invention.
[0128] Kits
[0129] In carrying out various assay, diagnostic, and therapeutic
methods of the invention, it is desirable to prepare in advance
kits comprises a combination of an anti-ROR-1 antibody described
herein with other materials. For example, in the case of sandwich
enzyme immunoassays, kits of the invention may contain a monoclonal
antibody that specifically binds ROR-1 optionally linked to an
appropriate carrier, a freeze-dried preparation or a solution of an
enzyme-labeled monoclonal antibody which can bind to the same
antigen together with the monoclonal antibody or of a polyclonal
antibody labeled with the enzyme in the same manner, a standard
solution of purified ROR-1, a buffer solution, a washing solution,
pipettes, a reaction container and the like. In addition, the kits
optionally include labeling and/or instructional materials
providing directions (i.e., protocols) for the practice of the
methods described herein. While the instructional materials
typically comprise written or printed materials, they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated. Such media
include, but are not limited to electronic storage media (e.g.,
magnetic discs, tapes, cartridges, chips), optical media (e.g., CD
ROM), and the like. Such media may include addresses to internet
sites that provide such instructional materials.
[0130] Having described the invention in detail, it will be
apparent that modifications, variations, and equivalent embodiments
are possible without departing the scope of the invention defined
in the appended claims. Furthermore, it should be appreciated that
all examples in the present disclosure are provided as non-limiting
examples.
EXAMPLES
[0131] The following non-limiting examples are provided to further
illustrate the present invention. It should be appreciated by those
of skill in the art that the techniques disclosed in the examples
that follow represent approaches the inventors have found function
well in the practice of the invention, and thus can be considered
to constitute examples of modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments that are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
Example 1
Demonstration of Production of Anti-Adenovirus Antibody
[0132] Chronic lymphocytic leukemia (CLL) CLL cells were transduced
with replication-defective adenovirus encoding CD154 (Ad-CD154).
The seven patients of the study all had progressive intermediate or
high-risk CLL by the modified Rai criteria. All patients had
performance status of 0 to 2, life expectancy of more than 3
months, and normal renal, hepatic, and pulmonary function on study
entry. Ad-CD154 were prepared and transduced into CLL cells as
described in Wierda et al. (2000) Blood 96, 2917-24; and Cantwell
et al. (1996) Blood 88, 4676-83. 3-6.times.108 transduced CLL cells
were injected biweekly 5 times in 6 patients except for one
patient. Sera, collected sequentially during the studies from these
6 patients, was examined. Before treatment 4 out of 6 patients had
hypogammaglobulinemia and residual 2 patients also have relatively
low titers of immunoglobulins. After completion of therapy total
IgG and IgM were slightly increased. (IgG; 656.+-.297 to 940.+-.487
p=0.04, IgA; 72.+-.63 to 69.+-.61 p=0.4, IgM; 38.+-.21 to 74.+-.48
p=0.07) (FIG. 1a).
[0133] The antibody response was measured against the recombinant
adenovirus used to transduce the CLL cells. Five of six patients
had a vigorous polyclonal antibody response to adenovirus antigens
following treatment (FIG. 1b). This response initially involved
antibodies of the IgM class, and then subsequently antibodies of
the IgG and IgA classes, but not IgE (FIG. 1c and not shown). On
average, 50-fold, 60-fold, or nearly 1,000-fold increases in the
titers of IgM, IgA, or IgG anti-adenovirus antibodies were
observed, respectively. The IgG response involved antibodies of
IgG1 and IgG3 isotypes (FIG. 1c), which primarily are observed in
Th1-type immune responses. Moreover, no significant increases were
observed in anti-adenovirus antibodies of the IgG4 isotype (data
not shown), which typically are observed in Th2-type immune
responses. In compared with this vigorous response, the increases
in the titers of anti-tetanus toxin antibodies were not obvious
unless patients received subsequent booster immunizations with
tetanus toxoid. In addition, development of autoantibodies to red
cells, platelets, or the human CD154 molecule following treatment
was not observed.
Example 2
Flow Cytometry Analysis of Anti-CLL Activities
[0134] Anti-CLL activities were determined by flow cytometry.
Peripheral blood mononuclear cells (PBMC) from IgG negative CLL
case or healthy donor were incubated with one-fifth diluted serum
from the patient or healthy donor, and bound IgG was detected by
mouse anti-human IgG antibody (Pharmingn). B cells (CD19+CD3-) were
gated using anti-CD19 antibodies conjugated APC and anti-CD3
antibody conjugated with FITC.
Example 3
ROR-1 Anti-Sera Production
[0135] Anti-ROR-1 mouse sera by means of DNA vaccination with ROR-1
expression vector. Eight-week old Balb/c female mice were injected
intradermally with 100 .mu.g of ROR-1 cDNA (Origene) with 50 .mu.g
of GM-CSF and CD154 expression vector as adjuvants. After 3 courses
of injection, sera was collected from the mice. Chinese hamster
ovary cell (CHO) with or without transfection with ROR-1 cDNA
cloned into pcDNA3 vector by lipofectamine 2000 (Invitrogen) was
used to determine the titer of anti-ROR-1 antibody in serum. Bound
antibody from immunized mice was detected by flow cytometry using
anti-mouse antibody with fluorescence (Pharmingen). To distinguish
the untransfected CHO from transfectants in the mixture, it was
stained with PKH26 (Sigma) according to the manufacture's protocol.
Anti-ROR-1 activity was determined by incubating CHO transfectants
and serum from patient followed by detection with anti-human Ig
labeled with fluorescence (Southern Biotech).
Example 4
ELISA
[0136] To produce recombinant ROR-1 protein, its extracellular
region was cloned into the pcDNA3-zeocin vector encoding rabbit IgG
Fc region in frame. Stable CHO transfectant (CHO-ROR-1rIg) was made
with this vector, and was adapted to suspension culture using IMGX
II medium (HyClone). Suspended CHO-ROR-1rIg was cultured in
ProCHO-5 medium, and recombinant ROR-1rIg was purified using
protein A sepharose (Pierce).
[0137] 5 .mu.g/ml protein or 10.sup.8/ml adenovirus was absorbed 96
well plate overnight at 4.degree. C. After washing and blocking
with 2% BSA/PBS, serum dilutions were added and incubated for 1
hour at room temperature. Goat anti-human Ig, IgG, IgA, IgM, IgG1,
IgG2, IgG3 and IgG4 conjugated with horseradish peroxidase (HRP) or
alkaline phosphatase (AP) (Southern Biotechnology, Birmingham,
Ala.) were used as secondary antibody. TMB (KPL, Gaithersburg, Md.)
or pNPP (Sigma) was used for substrate for HRP and AP respectively.
All experiments were done duplicate and were shown the average.
Example 5
Analysis of Microarray Data
[0138] Gene expression profiles of normal human tissues were
obtained from the data series GSE803 of Gene Expression Omnibus
(GEO) database. The gene set of CLL signature genes were made
according to the published papers Klein et al. (2001) J Exp Med
194, 1625-38; Rosenwald et al. (2001) J Exp Med 194, 1639-47. The
data were clustered and visualized with GeneSpring software
(Silicon Genetics).
Example 6
Immunoblotting
[0139] Total cell lysates were made by incubation cells in a lysis
buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 100 mM
NaCl, 50 mM NaF, 5 mM EDTA, 40 mM glycerophosphate, 1 mM sodium
orthovanadate, with complete protease inhibitor mix (Roche). Cell
lysates were separated 7.5% or 5-15% gradient SDS-PAGE and blotted
on Immobilon-P membrane (Millipore). For immunoblot, rabbit (Cell
signaling) or goat (R&D) anti-ROR-1 antibodies were used
followed with anti-rabbit or anti-goat antibodies conjugated with
HRP (Santa Cruz). Conditioned medium of culture with CHO with or
without transfection with HA-tagged Wnt5a cDNA (Upstate) was
incubated with 1 .mu.g of ROR-1rIg or rabbit IgG followed by
immunoprecipitation with anti-HA matrix (Roche) or protein A/G
agarose (SantaCruz). Bound proteins were immunoblotted with anti-HA
(Roche) or anti-rabbit Ig antibody.
Example 7
Reporter Assay
[0140] A reporter assay was performed as described in Lu et al.
(2004) Proc Natl Acad Sci USA 101, 3118-23. Briefly, HEK293 cells
were transfected in 12-well plates by using FuGENE (Roche,
Mannheim, Germany), and 0.5 .mu.g of reporter plasmid, 0.1-0.2
.mu.g of the control plasmid pCMX.beta.-gal, 100-200 ng of the
various expression plasmids, and carrier DNA pBluescriptKSII, for a
total of 1 .mu.g per well. The luciferase values were normalized
for variations in transfection efficiency by using the
.beta.-galactosidase internal control, and are expressed as fold
stimulation of luciferase activity, compared with the designated
control cultures. All of the transfection results are
representative of a minimum of three independent transfections.
Example 8
Induction of Humoral Immunity Against CLL Cell
[0141] The production of anti-adenovirus antibody suggests the
induction of humoral immunity against the CLL cell itself.
Allogeneic CLL cells were incubated with serum from patient before
and after treatment. Antibody binding was checked by flow
cytometry.
[0142] Results showed that the sera from 3 patients after Ad-CD154
therapy had the reactivity against CLL B cells compared with the
sera before therapy (FIG. 2a). The shift of the histograms were
reproducible with another 3 CLL B cells, and it was not detectable
against B cells from healthy donors (FIG. 2b). This data suggests a
TAA(s) may exist on the surface of CLL cells in a hidden fashion
from surveillance of immunity, becoming immunogenic after CLL
received the immune-costimulatory molecules.
[0143] The microarray analyses of CLL samples identified the
relatively small number of genes that are differentially expressed
in CLL cells in compared with normal B cell subsets and another
types of B cell malignancies. Klein et al. (2001) J Exp Med 194,
1625-38; Rosenwald et al. (2001) J Exp Med 194, 1639-47. These CLL
signature genes are candidates for TAAs of CLL. The expressions of
these genes were examined in normal human tissues because where
there is an abundant expression in normal tissue, antibody
production against such a gene cannot occur in vivo. The expression
profiles of CLL signature genes in normal adult tissues wad
determined (data not shown). Genes that had low expressions in all
tissues were spotlighted. Attention was directed to receptor
tyrosine kinase ROR-1 gene, because it is a probable cell surface
molecule and is expressed mainly in developing cells. Yoda et al.
(2003) J Recept Signal Transduct Res 23, 1-15; Al-Shawi et al.
(2001) Dev Genes Evol 211, 161-71; Matsuda et al. (2001) Mech Dev
105, 153-6 (2001).
Example 9
Immunoblot
[0144] FIG. 3 depicts an immunoblot demonstrating that the
anti-Ror1 mAb (designated 4A5) can immune precipitate the Ror1
protein from cells made to express human Ror1 (e.g. Chinese Hamster
Ovary (CHO)) cells or chronic lymphocytic leukemia (CLL) cells.
Prior antibodies to Ror1 were not mAbs, were generated against
peptides to Ror1, are of low affinity, and cannot immune
precipitate the Ror1 protein. As such, the 4A5 mAb can be used to
detect and/or isolate the Ror1 protein, which could have
diagnostic, treatment, and/or investigative value.
Example 10
ROR-1 Expression in CLL B Cells
[0145] To confirm the ROR-1 protein expression in CLL B cells,
immunoblot analysis, as described above, using anti-ROR-1 antibody
was performed. Results showed that the bands at the level of 128 kD
were detected in peripheral blood or splenocytes from CLL patients
(FIG. 4a). The size is compatible with the reported murine ROR-1
and bigger than deduced size of 101 kD from amino acid sequence
without putative leader sequence probably due to the
glycosylation29. This band could be detected neither in samples of
peripheral blood from healthy donor nor splenocytes from idiopathic
thrombocytopenia purpura patient. ROR-1 protein was detectable also
in some Burkitt's B cell lines at the same molecular weight (FIG.
4b).
Example 11
Cell Surface Localization of ROR-1 Protein in CLL B Cells
[0146] Cell surface localization of ROR-1 protein in CLL B cells
was confirmed via flow cytometry. Anti-ROR-1 mouse sera, produced
by means of DNA vaccination with ROR-1 expression vector, as
described above, was reacted with CHO transfected with ROR-1
(CHO-ROR-1) but not with CHO parental cell (FIG. 4c). Using this
anti-serum, ROR-1 expression was detected on cell surface of all
CLL samples examined (n=8) but not on PBMC from healthy donors
(n=3) (FIG. 4d).
Example 12
Induction of Anti-ROR-1 Antibody by Ad-CD154 Therapy
[0147] To confirm that the antibody against ROR-1 is included in
the antibodies against CLL cells induced by Ad-CD154 therapy, the
sera from patients was reacted with CHO and CHO-ROR-1 shown as FIG.
4c. Results showed that although serum from healthy donor or
patient before treatment contained same reactivity against CHO and
CHO-ROR-1, sera from patient after Ad-CD154 therapy contained more
Ig reacted with CHO-ROR-1 than with CHO (FIGS. 5a and b).
[0148] Further verification of the induction of anti-ROR-1 antibody
by Ad-CD154 therapy was established with an ELISA assay using the
recombinant extracellular domain of ROR-1 fused with rabbit IgG Fc
(FIG. 6a). Results showed that anti-ROR-1 antibody was clearly
identified in 4 patients (#2,5,6,7) after Ad-CD154 therapy. The
remaining one patient (#3) also had a weak anti-ROR-1 reaction
although one patient (#4) did not get anti-ROR-1 antibody by this
therapy. This #4 patient was profound hypogammaglobulinemia and was
totally unresponsive to this therapy with no decrease of white
blood cell count (data not shown). Thus, all responsive patient to
Ad-CD154 had induction of anti-ROR-1 antibody after completion of
therapy. In these patients, anti-ROR-1 antibody was not obvious
before Ad-CD154 therapy. Although three patients (#5,6,7) had some
reactivity also against rabbit IgG, this reactivity was also
detected before therapy (FIG. 6c). Collectively ROR-1 was expressed
on CLL B cells restrictedly and could induce humoral immunity by
means of immune-gene therapy.
Example 13
ROR-1 Activation of Intracellular Machinery Associated with
Development and Progression of CLL
[0149] To demonstrate that ROR-1 can activate intracellular
machinery associated with development or progression of CLL, the
influence of exogenous ROR-1 expression on the reporter gene
regulating various transcription factors in HEK293 cells was
examined. Various Wnt family members were co-transfected, as ROR-1
has a cystein-rich domain, which is shared between frizzled
receptors and can bind with Wnt family members.
[0150] Results showed that the expression of ROR-1 with any Wnt
factor did not activate T-cell transcription factor (TCF) (FIG. 7a,
data not shown), suggesting that ROR-1 does not signal via the
canonical Wnt-signaling pathway. ROR-1 could not activate nuclear
factors of activated T cells (NFAT), or AP-1 dependent gene
expression (FIG. 7a). However, it was observed that co-expression
of ROR-1 in HEK293 cells with Wnt5a, but not with any other Wnt
factor, induced activation of NF-.kappa.B (FIG. 7b). Induction of
NF-.kappa.B was dose dependent on expression of ROR-1 and Wnt5a,
but independent of expression of LPR5/6 that ordinarily serve as
co-receptors for the frizzled family of Wnt receptors (data not
shown). Recombinant extracellular region of ROR-1 could bind with
Wnt5a in vitro (FIG. 7c). This data suggests non-canonical Wnt
member, Wnt5a may be the ligand of ROR-1 and induce the activation
signaling in cells.
Example 14
Lymphoma Cell Isolation and Purification
[0151] Staining of CLL Cells from Patients #1, 2, or 3 with 4A5
mAb
[0152] As depicted in FIG. 8, the number of the CLL patient is
indicated at the left-hand margin. Each panel depicts the staining
of CLL with Alexa-647-conjugated 4A5 mAb (blue histogram) versus an
Alexa-647-conjugated isotype control mAb (red histograms). In the
first column is the staining of total peripheral blood mononuclear
cells, in the middle column is the staining of the CD19+(total B
cells), and in the far right column is the staining of cells that
express both CD19 and CD5 (CLL cells), indicated at the columns'
bottoms.
[0153] Staining of Cells from Normal Donors #1, 2, or 3 with 4A5
mAb
[0154] As depicted in FIG. 9, the number of the normal donor (NORM)
is indicated at the left-hand margin. Each panel depicts the
staining of cells with Alexa-647-conjugated 4A5 mAb (blue
histogram) versus an Alexa-647-conjugated isotype control mAb (red
histograms). In the first column is the staining of total
peripheral blood mononuclear cells, in the middle column is the
staining of the CD19+ (total B cells), and in the far right column
is the staining of cells that express both CD19 and CD5, as
indicated at the bottom of each column.
[0155] Staining of Cells from an Exceptional Normal Donors
[0156] Recent studies indicate that close to 4% of adults over the
age of 40 might have low numbers of cells similar to CLL cells in
the peripheral blood. Moreover, over 11% of normal donors who have
first degree relatives with CLL might have such cells in the
peripheral blood. In FIG. 10, it is shown that anti-Ror1 mAb 4A5
can detect an occasional normal donor with Ror1 positive cells.
Each panel depicts the staining of cells with Alexa-647-conjugated
4A5 mAb (blue histogram) versus an Alexa-647-conjugated isotype
control mAb (red histograms). In the first column is the staining
of total peripheral blood mononuclear cells, in the middle column
is the staining of the CD19+ (total B cells), and in the far right
column is the staining of cells that express both CD19 and CD5, as
indicated at the bottom of each column. As can be noted from this
figure, the Ror1 positive cells co-express CD5 and CD19, a
phenotype common with CLL cells.
[0157] Staining of CLL Cells in the Marrow
[0158] In FIG. 11, numbers corresponding to a CLL patient are
provided at the left-hand margin. Each panel depicts the staining
of cells with Alexa-647-conjugated 4A5 mAb (blue histogram) versus
an Alexa-647-conjugated isotype control mAb (red histograms). In
the first column is the staining of total marrow mononuclear cells,
in the middle column is the staining of the CD19+ (total B cells),
and in the far right column is the staining of cells that express
both CD19 and CD5 (CLL cells), as indicated at the bottom of each
column.
[0159] Staining of CLL Cells in the Marrow
[0160] The proportion of cells that express Ror1, as detected by
the mAb 4A5, are indicated in FIG. 12. Each dot represents the
proportion of cells from a single donor. The percent of cells
scoring positive is indicated by the y-axis. The left hand panel
provides the percent lymphocytes (as per light scatter) that stain
with 4A5 mAb. The right panel provides the percent of CD5+CD19+ B
cells that stain with 4A5. The left panel provides the percent of
lymphocytes that stain with 4A5 in samples obtained from the blood
normal donors (far left), the marrow of patients with CLL (middle),
or blood of patients with CLL (far right).
Example 15
Magnetic Bead Detection and Isolation of Lymphoma Cells
[0161] Lymphoma cells can be isolated and purified using the
following procedure: [0162] 1. Stain CLL cells with PKH67 [0163] 2.
Titrate CLL cells in normal PBMCs (10% to 0.1%) [0164] 3. Stain
cells with: [0165] a. Iso-Alexa647, CD5, CD19 [0166] b.
4A5-Alexa647, CD5, CD19 [0167] 4. Incubate for 20 min on ice
followed by a wash 2.times. with PBS-0.5% BSA [0168] 5. Add
magnetic beads(Miltenyi)to cells; Incubate 15 min on ice; Wash
1.times. with PBS-0.5% BSA) [0169] 6. Add column to magnet; Wash
1.times. with 3 ml PBS-0.5% BSA [0170] 7. Add this mixture to pre
washed column; Wash unbound cells 3.times. with 3 ml PBS-0.5% BSA;
(unbound fraction=4A5 NEG) [0171] 8. Remove column from magnet; Add
5 ml PBS-0.5% BSA; (bound fraction=4A5 POS)
[0172] Detection of CLL Cells Admixed with Normal Lymphocytes
[0173] 4A5+ CLL cells admixed with the lymphocytes from normal
donors are shown in FIG. 13. CLL cells were first stained with
PKH67, which labeled them bright green (as observed on the x axis),
allowing for their detection after being admixed with normal
lymphocytes. The stained CLL cells were mixed with the lymphocytes
of a normal donor and then the mixture was stained with an
Alexa-647-conjugated isotype control mAb (ISO) Alexa-647-conjugated
4A5, allowing for detection of the red fluorescence seen on the
y-axis.
[0174] Each panel represents a different mixture of cells stained
with either the isotype control mAb or 4A5, as indicated in the
key, which refers to the number in each panel of the figure. Those
samples stained with the isotype control mAb are indicated by the
term "Iso", those samples stained with 4A5 are indicated. The
percent preceding the CLL is the percent at which the CLL cells are
represented in the mixture. As seen from this figure, the 4A5 mAb
does not stain normal lymphocytes, allowing for detection of minute
proportions of CLL cells that are labeled green.
[0175] Isolation of CLL Cells Admixed with Normal Lymphocytes
[0176] Isolated 4A5+ CLL cells admixed with the lymphocytes from
normal donors are indicated in FIG. 14. CLL cells were stained,
mixed with normal lymphocytes at various ratios, and then stained
with fluorochrome-conjugated 4A5 mAb, as in Slide #6. Each panel
represents analyses of cells isolated from different mixtures of
CLL cells with normal lymphocytes, as indicated in the key, which
refers to the number in each panel of the figure. The percent
preceding the CLL is the percent at which the CLL cells are
represented in the mixture.
[0177] As seen from this figure, the 4A5 mAb does not stain normal
lymphocytes, allowing for detection of minute proportions of CLL
cells that are labeled green. As can be seen in these panels, this
method can isolate fairly pure populations of CLL cells from
mixtures of CLL cells with normal lymphocytes in which the CLL
cells constitute only a small fraction of the total cells.
Example 16
Detection of ROR-1 Antibody in Cancer but not Normal Cells
[0178] To evaluate ROR-1 surface expression in the human cell lines
listed in the Table below (Table 1), CHO cells were used as a
negative control and CHO-ROR-1 cells as a positive control for flow
cytometry. The ROR-1 antibody was the 4A5 mAb. The control mAb was
a conjugated isotype IgG2b mAb.
[0179] As shown in FIG. 15, the following cell lines stained
brightly with the 4A5 mAb, confirming ROR-1 expression: EW536, CLL,
786-0, HCT116, HT29, SW620, MDA-MB-231, MDA-MB-431, and MDA-MB-468.
CHO, MOLT-4, SW948, MCF-7, and SKBR3 were negative for ROR-1
expression, indicating preferential ROR-1 expression in this
population among adenocarcinoma and lymphoma.
[0180] In immunoblot studies of the same adult cancer tissues
(using a ROR-1 antibody raised against ROR-1 peptide), the same
cancer tissues reacted to indicate ROR-1 expression (FIGS. 16A and
16B). Using the A5A mAb, immuno precipitation studies confirmed
that ROR-1 was not found on normal tonsil cells of CHO, but is
strongly expressed in CLL, B cell lymphoma, and breast
adenocarcinomas, less so in colon adenocarcinoma (FIGS. 16C and
16D).
Example 17
ROR-1 DNA Vaccine Constructs
[0181] Vectors were constructed to encode the chimeric ROR1 protein
with ubiquitin located at the amino terminus separated from ROR1 by
an intervening codon for Met, and a separate vector with a codon
for the destabilizing amino acid Arg and an in-frame insert of a
segment of lacI. This segment contains a lysine residue spaced
optimally from the N-terminus. To generate the constructs, ROR1 was
PCR amplified from the pCMV6-XL-ROR1 vector (Origene) using primers
that encoded for NotI and XbaI. The PCR product was gel-purified,
cut with those restriction enzymes and ligated into a pcDNA3
subclone that contained the chimeric Ub-M-(lacI) or Ub-R-(lacI).
The final construct contains ROR1 3' of these sequences: Ub-M-ROR1
and Ub-R-ROR1.
[0182] Confirmation of ROR-1 Protein Expression by Cell-Free
Assay:
[0183] The constructs were evaluated for their capacity to direct
synthesis of the ROR-1 protein. For this in vitro transcription and
translation was performed using the TNT Quick coupled
Transcription/Translation System from Promega in the presence of
biotinylated lysine-specific tRNA. A luciferase plasmid served as
positive control for the reaction. Both constructs allowed for the
expression of one predominant protein at the size of ROR-1.
[0184] To demonstrate ROR-1 protein expression in mammalian cells,
P815 cells were transfected with Ub-M-ROR1 or Ub-R-ROR1 using the
Amaxa transfection system according to manufacturer's instructions
using program L13. The generation of such cells is described
below.
[0185] Generation of P815 Cells Expressing ROR-1
[0186] To examine the magnitude of the immune response generated by
the Ub-M-ROR-1 and Ub-R-ROR-1 DNA vaccine, CTL activity of
splenocytes harvested from immunized mice will be assessed against
the H-2d mastocytoma, P815, and P815 cells transfected to express
human ROR-1. To generate P815 cells that stably expressed ROR1,
P815 cells were transfected with pcDNA3-ROR-1, generated using the
Amaxa transfection system. To select ROR-1 expressing cells, the
cells were grown in G418 (400 .mu.g/ml). Subsequently the cells
were sub-cloned by limited dilation and analyzed for ROR-1
expression by flow cytometry. A stable P185 clone was generated
that expresses ROR-1 (FIG. 17; P815-ROR-1). This cell line will
serve as target for CTL assays.
[0187] To generate stable transfectants, the cells were
subsequently cultured under selection pressure in the presence of
400 .mu.g/ml G418. G418-resistant cells were cloned by limiting
dilution. To evaluate the relative intracellular stability of the
transgene products in the transfected P815 cells, cells were
cultured in the presence of a 26S proteasome inhibitor. P815 cells,
and P815 cells stably transfected with the Ub-M-ROR1 or Ub-R--ROR1
constructs were incubated in 100 .mu.M of the proteasome inhibitor
LLnL (N-acetyl-L-leucinyl-L-leucinal-L-norleucinal) for 18 h.
Lysates were prepared from the transfected cells and evaluated by
Immunoblot for ROR1 expression.
[0188] As expected, in the absence of LLnL, only P815 cells
transfected with the Ub-M-ROR-1, but not with Ub-R--ROR-1 expressed
detectable ROR-1 protein. Non-transfected P815 cells did not
express ROR-1. When both transfectants were cultured in the
presence of LLnL a strong increase in ROR-1 expression was
observed. These results show that ROR-1 expressed from the
Ub-M-ROR-1 and more so from the Ub-R--ROR-1 constructs was degraded
in the proteasome.
[0189] These constructs can be reasonably expected to induce
antibody responses or anti-ROR1 CTL responses. To this end, cell
based assays are useful to confirm the activity of candidate ROR-1
vaccines, to compare and contrast activity among candidates and
with ROR-1 constructs that are not targeted for degradation. CTL
activity is measurable using ROR1 expressing target cells and
target cells without ROR1 as controls; e.g., in the P815 cells
described. TABLE-US-00001 TABLE 1 Descriptions Of The Various Cell
Lines Used In FIG. 15 Name Source of Cells CHO Chinese hamster
ovary cells CHO-ROR1 CHO cells transfected to express human Ror1
EW36 Endemic African Burkitt's lymphoma (a B cell lymphoma) MOLT4
Human T cell lymphoma CLL Human chronic lymphocytic leukemia 786-0
Human renal cell carcinoma cell line HCT116 Human colon
adenocarcinoma cell line HT-29 Human colorectal adenocarcinoma cell
line SW620 Human colon adenocarcinoma cell line SW948 Human colon
cancer cell line MCF-7 Human, Caucasian, breast, adenocarcinoma
MDA-MB-231 Highly aggressive human, Caucasian, breast,
adenocarcinoma MDA-MB-431 Highly aggressive human, Caucasian,
breast, adenocarcinoma MDA-MB-468 Highly aggressive human,
Caucasian, breast, adenocarcinoma SKBR3 Human mammary carcinoma
* * * * *