U.S. patent application number 15/700457 was filed with the patent office on 2018-04-12 for chimeric rabbit/human ror1 antibodies.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Dept. of Health & Human Services. The applicant listed for this patent is The United States of America, as represented by the Secretary, Dept. of Health & Human Services, The United States of America, as represented by the Secretary, Dept. of Health & Human Services. Invention is credited to Christoph Rader, Jiahui Yang.
Application Number | 20180100020 15/700457 |
Document ID | / |
Family ID | 45099228 |
Filed Date | 2018-04-12 |
United States Patent
Application |
20180100020 |
Kind Code |
A1 |
Rader; Christoph ; et
al. |
April 12, 2018 |
CHIMERIC RABBIT/HUMAN ROR1 ANTIBODIES
Abstract
The invention relates to antibodies having specificity for human
ROR1, compositions thereof, and methods for using such antibodies,
including in the diagnosis and treatment of disorders associated
with aberrant ROR1 expression.
Inventors: |
Rader; Christoph; (Jupiter,
FL) ; Yang; Jiahui; (Kunming, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Dept. of Health & Human Services |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Dept. of Health & Human
Services
Bethesda
MD
|
Family ID: |
45099228 |
Appl. No.: |
15/700457 |
Filed: |
September 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13990977 |
Jun 7, 2013 |
9758586 |
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PCT/US11/62670 |
Nov 30, 2011 |
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15700457 |
|
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61418550 |
Dec 1, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/732 20130101;
C07K 2317/55 20130101; C07K 2317/92 20130101; C07K 16/2869
20130101; C07K 16/2857 20130101; C07K 2317/734 20130101; C07K
2317/624 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. An isolated antibody having specificity for the extracellular
domain of human receptor tyrosine kinase-like orphan receptor 1
(ROR1), wherein the antibody comprises: (a) a light chain variable
domain having at least 90% sequence identity to a sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID
NO: 5; or (b) a heavy chain variable domain having at least 90%
sequence identity to a sequence selected from the group consisting
of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6; or (c) both a
light chain of (a) and a heavy chain of (b).
2.-33. (canceled)
34. A method of killing or inhibiting the growth of cells
expressing ROR1 in a subject, which method comprises administering
a therapeutically effective amount of an isolated antibody of claim
1 to a subject in need thereof, thereby killing or inhibiting the
growth of cells expressing ROR1 in the subject.
35. A method of treating a disease or condition associated with
elevated expression of ROR1 in a subject, the method comprising
administering a therapeutically effective amount of an antibody of
claim 1 to a subject in need thereof, which subject has a disease
or condition associated with elevated expression of ROR1, thereby
treating the disease or condition associated with elevated
expression of ROR1 in the subject.
36. The method of claim 35, wherein the disease or condition is a
B-cell cancer, renal cell carcinoma, colon cancer, colorectal
cancer, or breast cancer.
37. The method of claim 36, wherein the disease or condition is
B-cell lymphocytic leukemia (B-CLL) or mantle cell lymphoma
(MCL).
38. The method of claim 34, wherein the antibody is selected from
the group consisting of an F(ab)2, Fv, scFv, IgG.DELTA.CH2,
F(ab')2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv,
(scFv)2, and a synthetic IgG.
39. The method of claim 38, wherein the antibody is conjugated to a
synthetic molecule.
40. The method of claim 39, wherein the antibody is a T-body and
the synthetic molecule comprises a transmembrane region and an
intracellular T-cell receptor (TCR) signaling domain.
41. The method of claim 39, wherein the synthetic molecule is a
cytotoxic agent, a radioisotope, or a liposome.
42. The method of claim 34, wherein the method further comprises
co-administering a pharmaceutical composition comprising a second
therapeutic agent to kill or inhibit the growth of cells expressing
ROR1 or to treat the disease or condition associated with elevated
expression of ROR1 in the subject.
43. A method of detecting an altered ROR1 level in a test sample,
which method comprises: (a) obtaining a test sample from a subject;
(b) contacting the sample with an isolated antibody of claim 1; (c)
determining the level of ROR1 in the test sample; and (d) comparing
the level of ROR1 in the test sample to a control level of ROR1 to
thereby determine whether the ROR1 level in the test sample is
altered relative to the control level of ROR1.
44. The method of claim 43, wherein a level of ROR1 in the subject
that is greater than the control level is indicative of a disease
or condition associated with elevated expression of ROR1 in the
subject.
45. The method of claim 44, wherein the disease or condition
associated with elevated expression of ROR1 is a B-cell cancer,
renal cell carcinoma, colon cancer, colorectal cancer, or breast
cancer.
46. The method of claim 44, wherein the disease or condition is
B-cell lymphocytic leukemia (B-CLL) or mantle cell lymphoma
(MCL).
47. A method of detecting an ROR1-expressing tumor in a subject,
which method comprises: (a) administering the antibody of claim 1
to a subject that has, is suspected to have, or is at risk for an
ROR1-expressing tumor; and (b) imaging the subject for a region of
altered conjugated label density or concentration, wherein the
density or concentration is relative to (i) background in proximal
tissue or (ii) the density or concentration previously detected in
the same region of the subject, such that the existence of a region
of altered conjugated label density or concentration is an
indication of the presence of an ROR1-expressing tumor in the
subject.
48. A method of detecting an ROR1-expressing B-cell tumor in a
subject, which method comprises: (a) administering the antibody of
claim 1 to a subject that has, is suspected to have, or is at risk
for a B-cell tumor; and (b) imaging the subject for a region of
altered conjugated label density or concentration, wherein the
density or concentration is relative to (i) background in proximal
tissue or (ii) the density or concentration previously detected in
the same region of the subject, such that the existence of a region
of altered conjugated label density or concentration is an
indication of the presence of an ROR1-expressing B-cell tumor in
the subject.
49. The method of claim 48, wherein the ROR1-expressing B-cell
tumor is a B-cell lymphocytic leukemia (B-CLL) or a mantle cell
lymphoma (MCL) tumor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 13/990,977, filed on Jun. 7, 2013, which is
the U.S. National Stage of PCT/US2011/062670, filed on Nov. 30,
2011, which claims the benefit of U.S. Provisional Patent
Application No. 61/418,550, filed Dec. 1, 2010, each of which is
incorporated by reference in its entirety herein.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 17,898 Byte
ASCII (Text) file named "729166_ST25.txt" created on Sep. 8,
2017.
BACKGROUND OF THE INVENTION
[0003] Antibody therapies and diagnostics have been developed for
use in treating a wide range of conditions including autoimmune
diseases or disorders, infectious diseases, and cancers. Such
therapies are useful but also can be associated with undesirable
immunogenicity and can damage healthy cells and tissues.
[0004] B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell
lymphoma (MCL) are two incurable B-cell malignancies with a
combined incidence of new cases that exceeds 18,000 patients per
year in the United States alone. Antibody therapies have been
developed for B cell malignancies, which include rituximab, a
chimeric mouse/human monoclonal antibody (mAb), alemtuzumab, a
humanized mAb, and ofatumumab, a human mAb. However, the target
antigens for all three of these drugs (CD20, CD52, and CD20
respectively) are expressed not only in malignant B cells but also
in normal B cells, and CD52 is ubiquitously expressed on a variety
of normal cells of the immune system. Therefore, immunosuppression
can be a concern with these antibody therapies. Currently in the
United States and Europe, there is no commercial therapeutic
antibody that specifically recognizes an antigen present on
malignant B cells, but not on normal B cells.
[0005] There is a desire for additional therapeutic and diagnostic
antibodies having good efficacy and that exhibit minimal binding
and/or damage to non-diseased cells.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention provides an isolated antibody with specificity
for the extracellular domain of receptor tyrosine kinase-like
orphan receptor 1 (ROR 1), which is selectively expressed on the
surface of malignant cells, including B-cell tumors and other
cancers.
[0007] In particular, the invention provides an isolated antibody
having specificity for human ROR1 and having (a) a light chain with
at least 90% identity to a sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, (b) a
heavy chain with at least 90% identity to a sequence selected from
the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:
6, or (c) both a light chain of (a) and a heavy chain of (b).
[0008] The invention additionally provides an isolated antibody
having specificity for human ROR1 and having (a) a light chain with
at least 90% identity to the sequence of SEQ ID NO: 1, (b) a heavy
chain with at least 90% identity to the sequence of SEQ ID NO: 2;
or (c) both a light chain of (a) and a heavy chain of (b).
[0009] The invention additionally provides an isolated antibody
having specificity for human ROR1 and having (a) a light chain with
at least 90% identity to the sequence of SEQ ID NO: 3, (b) a heavy
chain with at least 90% identity to the sequence of SEQ ID NO: 4;
or (c) both a light chain of (a) and a heavy chain of (b).
[0010] The invention further provides an isolated antibody having
specificity for human ROR1 and having (a) a light chain with at
least 90% identity to the sequence of SEQ ID NO: 5, (b) a heavy
chain with at least 90% identity to the sequence of SEQ ID NO: 6;
or (c) both a light chain of (a) and a heavy chain of (b).
[0011] The invention also provides an isolated antibody having
specificity for human ROR1 and having at least one CDR that
includes a sequence selected from the group consisting of SEQ ID
NOs: 31-48. In other embodiments, the isolated antibody can include
one or more variants of the foregoing CDRs with 1, 2, or 3 amino
acid substitutions, insertions, or deletions.
[0012] The invention further provides a pharmaceutical composition
comprising an antibody of the invention and a pharmaceutically
acceptable carrier.
[0013] In addition, the invention provides a method of killing or
inhibiting the growth of cells expressing ROR1 in a subject, as
well as a method of treating a disease or condition associated with
elevated expression of ROR1 (e.g., a B-cell malignancy, renal cell
carcinoma, colon cancer, or breast cancer), by administering a
therapeutically effective amount of an isolated antibody of the
invention or a pharmaceutical composition thereof to a subject in
need thereof, thereby killing or inhibiting the growth of cells
expressing ROR1 in the subject, or treating the disease or
condition associated with elevated expression of ROR1 in the
subject.
[0014] The antibodies and compositions of the invention also can be
used in diagnostic methods to detect altered levels of ROR1, e.g.,
in a sample or in a subject, or ROR1-expressing tumors in a
subject.
BRIEF DESCRIPTION OF THE DRAWING(S)
[0015] FIG. 1 is a schematic depiction of eight recombinant Fc
fusion proteins having different compositions of the
Immunoglobulin- (Ig-), Frizzled-, and Kringle-like extracellular
domains of ROR1. Ig domains are depicted as ovals (in black),
Frizzled domains are depicted as triangles (in black), and Kringle
domains are depicted as circles (in black).
[0016] FIG. 2 is a list of the amino acid sequences corresponding
to the R11, R12, and Y31 variable region light chains
(V.sub..kappa. and V.sub..lamda.) (SEQ ID NOs: 1, 3, and 5) and
heavy chains (V.sub.H) (SEQ ID NOs: 2, 4, and 6), which identify
light chain framework regions FR1-FR4 (SEQ ID NOs: 7-10, 11-14, and
15-18), light chain complementarity determining regions CDR1-CDR3
(SEQ ID NOs: 31-33, 34-36, and 37-39), heavy chain framework
regions FR1-FR4 (SEQ ID NOs: 19-22, 23-26, and 27-30), and heavy
chain CDR1-CDR3 (SEQ ID NOs: 40-42, 43-45, and 46-48).
[0017] FIG. 3A is a graph that depicts the results of ELISA
studies, providing absorbance data for binding of chimeric
rabbit/human IgG1 R11, R12, and Y31, and negative control P14,
against immobilized human ROR1 (Fc-hROR1), mouse ROR1 (Fc-mROR1),
and human ROR2 (hROR2-Fc). Columns indicate mean values, and error
bars indicate standard deviation values of triplicates.
[0018] FIG. 3B is a graph that depicts the results of ELISA studies
mapping the epitopes of IgG1 R11, R12, and Y31, and negative
control P14 with five immobilized Fc fusion proteins that consisted
of only one or two extracellular domains of human ROR1: Fc-hROR1ig
(ig), Fc-hROR1fz (fz), Fc-hROR1kr (kr), Fc-hROR1ig+fz (ig+fz), and
Fc-hROR1fz+kr (fz+kr). Columns indicate mean values, and error bars
indicate standard deviation values of triplicates.
[0019] FIG. 4 is a series of graphs that depict the results of
surface plasmon resonance binding analyses obtained for the binding
of IgG1 R11, R12, and Y31 to immobilized Fc-hROR1. Response unit (y
axis) increases that exceeded the values found for IgG1 R11, R12,
and Y31 alone indicated independent epitopes that allow
simultaneous binding. The x axis depicts the time in seconds
(s).
[0020] FIG. 5A is a series of graphs that depict the results of
surface plasmon resonance binding analysis obtained for the binding
of Fab and IgG1 R11, R12, and Y31 to immobilized Fc-hROR1 after
instantaneous background depletion. The mAbs were tested at five or
six different concentrations ranging from 1.5 to 100 nM. Each
concentration was tested in duplicate.
[0021] FIG. 5B is a series of graphs that depict the results of
surface plasmon resonance binding analysis obtained for the binding
of Fab and IgG1 R11 and Y31 to immobilized Fc-mROR1 after
instantaneous background depletion. The mAbs were tested at five or
six different concentrations ranging from 1.5 to 100 nM. Each
concentration was tested in duplicate.
[0022] FIG. 6A is a graph that depicts flow cytometry analysis of
the binding of IgG1 R11 (5 .mu.g/ml), R12 (1 .mu.g/ml), and Y31 (5
.mu.g/ml) to the surface of JeKo-1 cells. The gray shade indicates
the background observed with human anti-tetanus toxoid mAb TT11 in
IgG1 format (TT11) (5 .mu.g/ml). Biotinylated IgG1 was detected
with PE-streptavidin. They axis depicts the number of events, and
the x axis depicts the fluorescence intensity.
[0023] FIG. 6B is a graph that depicts flow cytometry analysis of
the binding of IgG1 R11 (5 .mu.g/mL), R12 (1 .mu.g/mL), and Y31 (5
.mu.g/mL) to the surface of HBL-2 cells. The gray shade indicates
the background observed with IgG1 TT11 (5 .mu.g/mL). Biotinylated
IgG1 was detected with PE-streptavidin. The y axis depicts the
number of events, and the x axis depicts the fluorescence
intensity.
[0024] FIG. 6C is a series of graphs that depict the results of
flow cytometry analysis of the binding of IgG1 R11 (5 .mu.g/ml),
R12 (1 .mu.g/ml), and Y31 (5 .mu.g/ml) to the surface of peripheral
blood mononuclear cells (PBMC) from chronic lymphocytic leukemia
(CLL) patients for CD19+CD5+ cells and CD19-CD5+ cells. The gray
shade indicates the background observed with negative control
chimeric rabbit/human IgG1 P14 (5 .mu.g/ml). Biotinylated IgG1 in
combination with FITC-CD19/APC-CD5 was detected with
phycoerythrin-streptavidin (PE-streptavidin). The y axis depicts
the number of events, and the x axis depicts the fluorescence
intensity.
[0025] FIG. 7A is a series of graphs that depict the results of
flow cytometry analysis of the binding of IgG1 R12 (5 .mu.g/ml),
R12 (1 .mu.g/ml), and Y31 (5 .mu.g/ml) to the surface of PBMC from
a CLL patient designated CLL-2 to identify PBMC subpopulations of
NK cells (CD16+CD3-), T cells (CD16-CD3+, CD19-CD5+), and CLL cells
(CD19+CD5+). The x and y axes in the top and middle rows depict
fluorescence intensity. In the bottom row, the gray shade indicates
the background observed with negative control PE-streptavidin
alone. The y axis depicts the number of events, and the x axis
depicts the fluorescence intensity.
[0026] FIG. 7B is a is a series of graphs that depict the results
of flow cytometry analysis of the binding of IgG1 R12 (5 .mu.g/ml),
R12 (1 .mu.g/ml), and Y31 (5 .mu.g/ml) to the surface of PBMC from
a CLL patient designated CLL-3 to identify PBMC subpopulations of
NK cells (CD16+CD3-), T cells (CD16-CD3+, CD19-CD5+), and CLL cells
(CD19+CD5+). The x and y axes in the top and middle rows depict
fluorescence intensity. In the bottom row, the gray shade indicates
the background observed with negative control PE-streptavidin
alone. The y axis depicts the number of events, and the x axis
depicts the fluorescence intensity.
[0027] FIG. 7C is a is a series of graphs that depict the results
of flow cytometry analysis of the binding of IgG1 R12 (5 .mu.g/ml),
R12 (1 .mu.g/ml), and Y31 (5 .mu.g/ml) to the surface of PBMC from
a CLL patient designated CLL-4 to identify PBMC subpopulations of
NK cells (CD16+CD3-), T cells (CD16-CD3+, CD19-CD5+), and CLL cells
(CD19+CD5+). The x and y axes in the top and middle rows depict
fluorescence intensity. In the bottom row, the gray shade indicates
the background observed with negative control PE-streptavidin
alone. The y axis depicts the number of events, and the x axis
depicts the fluorescence intensity.
[0028] FIG. 7D is a is a series of graphs that depict the results
of flow cytometry analysis of the binding of IgG1 R12 (5 .mu.g/ml),
R12 (1 .mu.g/ml), and Y31 (5 .mu.g/ml) to the surface of PBMC from
a CLL patient designated CLL-5 to identify PBMC subpopulations of
NK cells (CD16+CD3-), T cells (CD16-CD3+, CD19-CD5+), and CLL cells
(CD19+CD5+). The x and y axes in the top and middle rows depict
fluorescence intensity. In the bottom row, the gray shade indicates
the background observed with negative control PE-streptavidin
alone. The y axis depicts the number of events, and the x axis
depicts the fluorescence intensity.
[0029] FIG. 8A is a graph that depicts flow cytometry results of
IgG1 R11, R12, and Y31 in comparison to IgG1 P14 (negative
control), unspecific polyclonal human IgG (hIgG; negative control),
and rituximab (RTX; positive control) toward JeKo-1 cells in the
presence of rabbit complement. PI staining indicating cytotoxicity
was observed for rituximab only.
[0030] FIG. 8B is a graph that depicts flow cytometry results of
IgG1 R11, R12, and Y31 in comparison to IgG1 P14 (negative
control), unspecific polyclonal human IgG (hIgG; negative control),
and rituximab (RTX; positive control) toward HBL-2 cells in the
presence of rabbit complement. PI staining indicating cytotoxicity
was observed for rituximab only.
[0031] FIG. 8C is a graph that depicts flow cytometry results of
IgG1 R11, R12, and Y31 in comparison to IgG1 P14 (negative
control), unspecific polyclonal human IgG (hIgG; negative control),
and rituximab (RTX; positive control) toward PBMC from untreated
CLL patients in the presence of rabbit complement. PI staining
indicating cytotoxicity was observed for rituximab only.
[0032] FIG. 9A is a graph depicting results of a bioluminescent
intracellular protease detection assay quantifying the ADCC potency
of IgG1 R11, R12, and Y31 in comparison to human anti-tetanus
toxoid mAb TT11 in IgG1 format (negative control) and rituximab
(RTX; positive control) toward JeKo-1 cells and HBL-2 cells at a
concentration of 5 .mu.g/ml. Columns indicate mean values, and
error bars indicate standard deviation values of triplicates.
[0033] FIG. 9B is a graph depicting results of a cytotoxicity assay
against PBMC from three CLL patients, with mean values indicated by
horizontal bars.
[0034] FIG. 9C is a graph depicting results of a bioluminescent
intracellular protease detection assay quantifying the
antigen-dependent cellular cytotoxicity (ADCC) potency of IgG1 R11,
R12, and Y31 in comparison to human anti-tetanus toxoid mAb TT11 in
IgG1 format (negative control) and rituximab (RTX) toward HBL-2
cells at concentrations of 20 .mu.g/ml, 10 .mu.g/ml, 5 .mu.g/ml,
2.5 .mu.g/ml, 0.5 .mu.g/ml, 0.1 .mu.g/ml, and 0.02 .mu.g/ml, with
each concentration presented from left (black bars) to right (white
bars), respectively. Columns indicate mean values, and error bars
indicate standard deviation values of triplicates.
[0035] FIG. 10A is a series of graphs that depict the results of
flow cytometry analysis of human ROR1 cell surface densities on
primary CLL cells using biotinylated IgG1 R12 followed by
PE-streptavidin. The gray shade indicates the background observed
with negative control PE-streptavidin alone. The y axis depicts the
number of events, the x axis depicts the fluorescence intensity.
Mean fluorescence intensity (MFI) values are indicated.
[0036] FIG. 10B is a series of graphs that depict MFI reduction
over time of primary CLL cells reflecting the internalization of
IgG1 R11, R12, and Y31 into the cells in the absence or presence of
endocytosis inhibitor phenylarsine oxide (PAO).
[0037] FIG. 11A is a graph depicting apoptosis in PBMC from three
untreated CLL patients (CLL-2 (black bar), CLL-3 (dashed gray bar),
and CLL-4 (white bar)) cultured in the absence of fetal bovine
serum (FBS) and incubated with IgG1 R11, R12, Y31, or TT11, as
compared to positive control rituximab (RTX) and negative control,
and further in the presence (+) and absence (-) of F(ab').sub.2
goat-anti-human IgG (cross linker).
[0038] FIG. 11B is a graph depicting apoptosis in PBMC from three
untreated CLL patients (CLL-2 (black bar), CLL-3 (dashed gray bar),
and CLL-4 (white bar)) cultured in the presence of FBS and
incubated with IgG1 R11, R12, Y31, or TT11, as compared to positive
control rituximab (RTX) and negative control, and further in the
presence (+) and absence (-) of F(ab').sub.2 goat-anti-human IgG
(cross linker), as well as in the presence (+) and absence (-) of
IL-4 and CD40L.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a
conserved embryonic protein whose expression becomes progressively
reduced during embryonic development in mammals. The intact
protein, including its extracellular domain, does not appear to be
significantly expressed in normal, adult mammalian tissues. In
particular, studies have not identified significant expression of
ROR1 on the cell surface of normal adult human tissues, including
normal B cells. Baskar et al., Clin. Cancer Res., 14: 396-404
(2008), DaneshManesh et al., Int. J. Cancer, 123: 1190-1195 (2008),
and Fukuda et al., Proc. Nat'l. Acad. Sci. USA, 105: 3047-3052
(2008). However, ROR1 is expressed on the cell surface of malignant
B-cells, including B-cell chronic lymphocytic leukemia (B-CLL) and
mantle cell lymphoma (MCL). It has also been reported that ROR1 is
expressed in certain other cancer cell lines including Burkitt
lymphoma, renal cell carcinoma, colon cancer, and breast cancer.
See U.S. Patent Application Publication 2007/0207510. Therefore,
ROR1 can be considered a selective marker for these cancers. The
invention provides an antibody to this selective marker.
[0040] The invention provides an antibody having specificity for
ROR1, comprising (a) a light chain having at least 90% identity to
SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; (b) a heavy chain
variable domain having at least 90% sequence identity to SEQ ID NO:
2, SEQ ID NO: 4, or SEQ ID NO: 6; or (c) both a light chain of (a)
and a heavy chain of (b). In a preferred embodiment, the antibody
comprises both a light chain of (a) and a heavy chain of (b).
[0041] In one embodiment, the invention provides an antibody having
specificity for ROR1, comprising (a) a light chain having at least
90% identity to SEQ ID NO: 1; (b) a heavy chain variable domain
having at least 90% sequence identity to SEQ ID NO: 2; or (c) both
a light chain of (a) and a heavy chain of (b). In a preferred
embodiment, the antibody comprises both a light chain of (a) and a
heavy chain of (b).
[0042] In another embodiment, the invention provides an antibody
having specificity for ROR1, comprising (a) a light chain having at
least 90% identity to SEQ ID NO: 3; (b) a heavy chain variable
domain having at least 90% sequence identity to SEQ ID NO: 4; or
(c) both a light chain of (a) and a heavy chain of (b). In a
preferred embodiment, the antibody comprises both a light chain of
(a) and a heavy chain of (b).
[0043] In a further embodiment, the invention provides an antibody
having specificity for ROR1, comprising (a) a light chain having at
least 90% identity to SEQ ID NO: 5; (b) a heavy chain variable
domain having at least 90% sequence identity to SEQ ID NO: 6; or
(c) both a light chain of (a) and a heavy chain of (b). In a
preferred embodiment, the antibody comprises both a light chain of
(a) and a heavy chain of (b).
[0044] The antibody can be an isolated antibody having specificity
for human ROR1, wherein the antibody comprises a light chain having
at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:
5. In other embodiments, the percentage identity can be at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, or even
100%. In preferred embodiments, the light chain has at least 95%
identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In more
preferred embodiments, the light chain has 100% identity to SEQ ID
NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.
[0045] The antibody can be an isolated antibody having specificity
for human ROR1, wherein the antibody comprises a heavy chain having
at least 90% identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
6. In other embodiments, the percentage identity can be at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, or even
100%. In preferred embodiments, the heavy chain has at least 95%
identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In more
preferred embodiments, the heavy chain has 100% identity to SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
[0046] In some embodiments, the antibody can comprise any heavy
chain as described above, in combination with any suitable light
chain, such as those described above. Likewise, the antibody can
comprise any of the light chains as described above in combination
with any suitable heavy chain, such as those described above. For
example, in preferred embodiments, the antibody comprises a light
chain having at least 90% identity to SEQ ID NO: 1 and a heavy
chain having at least 90% identity to SEQ ID NO: 2, or a light
chain having at least 90% identity to SEQ ID NO: 3 and a heavy
chain having at least 90% identity to SEQ ID NO: 4, or a light
chain having at least 90% identity to SEQ ID NO: 5 and a heavy
chain having at least 90% identity to SEQ ID NO: 6. In a preferred
embodiment, the antibody comprises the light chain of SEQ ID NO: 1
and the heavy chain of SEQ ID NO: 2, the light chain of SEQ ID NO:
3 and the heavy chain of SEQ ID NO: 4, or the light chain of SEQ ID
NO: 5 and the heavy chain of SEQ ID NO: 6.
[0047] Percent (%) identity of peptide sequences can be calculated,
for example, as 100.times.[(identical positions)/min(TGA, TGB)],
where TGA and TGB are the sum of the number of residues and
internal gap positions in peptide sequences A and B in the
alignment that minimizes TGA and TGB. See, e.g., Russell et al., J.
Mol Biol., 244: 332-350 (1994).
[0048] The antibody of the invention can be any antibody including
a full length antibody or an antibody fragment having specificity
for the extracellular domain of human ROR1. For example, the
antibody can be polyclonal, monoclonal, recombinant, chimeric, or
humanized. Furthermore, the antibody can be of any isotype
including without limitation IgA, IgD, IgE, IgG, or IgM. Thus, for
example, the antibody can be any IgA such as IgA1 or IgA2, or any
IgG such as IgG1, IgG2, IgG3, IgG4, or synthetic IgG. The antibody
can also be any antibody fragment having specificity for the
extracellular domain of human ROR1, such as F(ab)2, Fv, scFv,
IgG.DELTA.CH2, F(ab')2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2,
dsFv, Fv, scFv-Fc, (scFv)2, a diabody, and a bivalent antibody. The
antibody can be any modified or synthetic antibody, including, but
not limited to, non-depleting IgG antibodies, T-bodies, or other Fc
or Fab variants of antibodies.
[0049] In addition to a heavy chain as described above, the
antibody of the invention can further comprise a light chain
selected from a Fab library using sequential naive chain shuffling.
Likewise, in addition to a light chain as described above, the
antibody of the invention can further comprise a heavy chain
selected from a Fab library using sequential naive chain
shuffling.
[0050] In some embodiments, the invention provides an isolated
antibody, having specificity for human ROR1, comprising at least
one CDR having a sequence selected from the group consisting of SEQ
ID NOs: 31-48. The invention also provides an isolated antibody
with specificity for ROR1 comprising at least one or more variants
of the foregoing CDR sequences, which include 1, 2, or 3
substitutions, insertions, deletions, or combinations thereof in a
sequence selected from the group consisting of SEQ ID NOs: 31-48.
For example, a recombinant chimeric or humanized antibody (or
fragment thereof) can include one, two, three, four, five, or six
of the foregoing CDR sequences. In preferred embodiments, however,
the recombinant chimeric or humanized antibody (or fragment
thereof) includes three CDR sequences of the same light or heavy
chain, e.g., SEQ ID NOs: 31-33, SEQ ID NOs: 34-36; SEQ ID NOs:
37-39; SEQ ID NOs: 40-42; SEQ ID NOs: 43-45; or SEQ ID NOs: 46-48.
In more preferred embodiments, the recombinant chimeric or
humanized antibody (or fragment thereof) includes six CDR sequences
of the same antibody, e.g., (a) SEQ ID NOs: 31-33 and SEQ ID NOs:
40-42; (b) SEQ ID NOs: 34-36 and SEQ ID NOs: 43-45; or (c) SEQ ID
NOs: 37-39 and SEQ ID NOs: 46-48.
[0051] In some embodiments, the invention provides an antibody with
avidity for ROR1 of about 10 .mu.M or less, 5 .mu.M or less, 2
.mu.M or less, 1 .mu.M or less, 500 nM or less, 400 nM or less, 300
nM or less, or 200 nM or less. The invention also provides an
antibody with avidity for ROR1 of about 100 nM or less, about 75 nM
or less, about 50 nM or less, about 25 nM or less, about 10 nM or
less, or about 5 nM or less. The invention further provides an
antibody with avidity for ROR1 of about 1 nM or less, about 800 pM
or less, about 700 pM or less, about 600 pM or less, about 500 pM
or less, about 400 pM or less, about 300 pM or less, about 200 pM
or less, or about 100 pM or less. Avidity can be measured using
art-known techniques, such as ELISA or surface plasmon
resonance.
[0052] The antibody of the invention can be produced by any
suitable technique, for example, using any suitable eukaryotic or
non-eukaryotic expression system. In certain embodiments, the
antibody is produced using a mammalian expression system.
[0053] The antibody of the invention can be produced using a
suitable non-eukaryotic expression system such as a bacterial
expression system. Bacterial expression systems can be used to
produce fragments such as a F(ab)2, Fv, scFv, IgG.DELTA.CH2,
F(ab')2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv,
scFv-Fc, (scFv)2, and diabodies. Techniques for altering DNA coding
sequences to produce such fragments are known in the art.
[0054] The antibody of the invention can be conjugated to a
synthetic molecule using any type of suitable conjugation.
Recombinant engineering and incorporated selenocysteine (e.g., as
described in International Patent Application Publication
WO/2008/122039) can be used to conjugate a synthetic molecule.
Other methods of conjugation can include covalent coupling to
native or engineered lysine side-chain amines or cysteine
side-chain thiols. See, e.g., Wu et al., Nat. Biotechnol., 23:
1137-1146 (2005). The synthetic molecule can be any molecule such
as one targeting a tumor. Of course, it will be understood that the
synthetic molecule also can be a protein (e.g., an antibody) or an
RNA or DNA aptamer.
[0055] Synthetic molecules include therapeutic agents such as
cytotoxic, cytostatic, or antiangiogenic agents, radioisotopes, and
liposomes. A cytotoxic agent can be a plant, fungal, or bacterial
molecule (e.g., a protein toxin). A therapeutic agent can be a
maytansinoid (e.g., maytansinol or DM1 maytansinoid), a taxane, a
calicheamicin, a cemadotin, or a monomethylauristatin (e.g.,
monomethylauristatin E or monomethylauristatin F). Therapeutic
agents include vincristine and prednisone. A therapeutic agent can
be an antimetabolite (e.g., an antifolate such as methotrexate, a
fluoropyrimidine such as 5-fluorouracil, cytosine arabinoside, or
an analogue of purine or adenosine); an intercalating agent (for
example, an anthracycline such as doxorubicin, daunomycin,
epirubicin, idarubicin, mitomycin-C, dactinomycin, or mithramycin);
a platinum derivative (e.g., cisplatin or carboplatin); an
alkylating agent (e.g., nitrogen mustard, melphalan, chlorambucil,
busulphan, cyclophosphamide, ifosfamide nitrosoureas or thiotepa);
an antimitotic agent (e.g., a vinca alkaloid such as vincristine,
or a taxoid such as paclitaxel or docetaxel); a topoisomerase
inhibitor (for example, etoposide, teniposide, amsacrine,
topotecan); a cell cycle inhibitor (for example, a flavopyridol);
or a microbtubule agent (e.g., an epothilone, discodermolide
analog, or eleutherobin analog). A therapeutic agent can be a
proteosome inhibitor or a topoisomerase inhibitor such as
bortezomib, amsacrine, etoposide, etoposide phosphate, teniposide,
or doxorubicin. Therapeutic radioisotopes include iodine
(.sup.131I), yttrium (.sup.90Y), lutetium (.sup.177Lu), actinium
(.sup.225Ac), praseodymium, astatine (.sup.211At), rhenium
(.sup.186Re), bismuth (.sup.212Bi or .sup.213Bi), and rhodium
(.sup.188Rh). Antiangiogenic agents include linomide, bevacuzimab,
angiostatin, and razoxane. The synthetic molecule can be another
antibody such as rituximab or bevacuzimab.
[0056] A synthetic molecule can also be a label. Labels can be
useful in diagnostic applications and can include, for example,
contrast agents. A contrast agent can be a radioisotope label such
as iodine (.sup.131I or .sup.125I), indium (.sup.111In), technetium
(.sup.99Tc), phosphorus (.sup.32P), carbon (.sup.14C), tritium
(.sup.3H), other radioisotope (e.g., a radioactive ion), or a
therapeutic radioisotope such as one of the therapeutic
radioisotopes listed above. Additionally, contrast agents can
include radiopaque materials, magnetic resonance imaging (MRI)
agents, ultrasound imaging agents, and any other contrast agents
suitable for detection by a device that images an animal body. A
synthetic molecule can also be a fluorescent label, a biologically
active enzyme label, a luminescent label, or a chromophore
label.
[0057] In yet other embodiments, the synthetic molecule can be a
liposome, as described in Bendas, BioDrugs, 15(4): 215-224 (2001).
In such embodiments, the antibody can be conjugated to a colloidal
particle, e.g., a liposome, and used for controlled delivery of an
agent to diseased cells. In preparing an antibody conjugated to a
liposome, e.g., an immunoliposome, an agent such as a
chemotherapeutic or other drug can be entrapped in the liposome for
delivery to a target cell.
[0058] In some embodiments, the antibody can also have specificity
for one or more antigens in addition to ROR1. For example, the
antibody of the invention can be engineered (e.g., as a bivalent
diabody or a conjugated Fab dimer or trimer) to have specificity
for ROR1 and another tumor antigen, e.g., an antigen associated
with B-CLL, MCL, Burkitt lymphoma, renal cell carcinoma, colon
cancer (e.g., colon adenocarcinoma), or breast cancer (e.g., breast
adenocarcinoma). The antibody can be engineered to have specificity
for ROR1 and an antigen that promotes activation or targeting of
cytotoxic effector cells.
[0059] The invention further provides eukaryotic or non-eukaryotic
cells that have been recombinantly engineered to produce an
antibody of the invention. The eukaryotic or non-eukaryotic cells
can be used as an expression system to produce the antibody of the
invention. In another embodiment, the invention provides ROR1
targeted immune cells that are engineered to recombinantly express
an ROR1 specific antibody of the invention. For example, the
invention provides a T-cell engineered to express an antibody of
the invention (e.g., an scFv, scFv-Fc, or (scFv)2), which is linked
to a synthetic molecule with the following domains: a spacer or
hinge region (e.g., a CD28 sequence or a IgG4 hinge-Fc sequence), a
transmembrane region (e.g., a transmembrane canonical domain), and
an intracellular T-cell receptor (TCR) signaling domain, thereby
forming a T-body (or chimeric antigen receptor (CAR)).
Intracellular TCR signaling domains that can be included in a
T-body (or CAR) include, but are not limited to, CD3.zeta.,
FcR-.gamma., and Syk-PTK signaling domains as well as the CD28,
4-1BB, and CD134 co-signaling domains. Methods for constructing
T-cells expressing a T-body (or CAR) are known in the art. See,
e.g., Marcu-Malina et al., Expert Opinion on Biological Therapy,
Vol. 9, No. 5 (posted online on Apr. 16, 2009).
[0060] The invention provides a method of inhibiting cells that
express ROR1 (ROR1 cells) by contacting the cells with an antibody
of the invention. The antibody can be a naked (unconjugated)
antibody or an antibody conjugated to a synthetic molecule, e.g., a
cytotoxic, cytostatic, or antiangiogenic agent, a radioisotope, or
even to a liposome. The method can be used to inhibit ROR1 cells in
vitro or in a subject (i.e., in vivo). The contacted ROR1 cells can
be in, for example, a cell culture or animal model of a disorder
associated with elevated levels of ROR1. The method is useful, for
example, to measure and/or rank (relative to another antibody) the
antibody's inhibitory activity for a specific ROR1 cell type.
Inhibiting ROR1 cells can include blocking or reducing the activity
or growth of ROR1 cells. Inhibiting can also include the killing of
ROR1 cells. While the method is not bound by or limited to any
particular mechanism of action, inhibitory activity can be mediated
by blocking ROR1-mediated signaling or by blocking the signaling of
an ROR1 associated receptor. Inhibitory activity can also be
mediated by recruitment of immune system effectors that attack ROR1
cells, e.g., by activating constituents of the antibody-dependent
cell-mediated cytotoxicity (ADCC) or complement systems.
[0061] The invention also provides a method of treating a subject
that has, is suspected to have, or is at risk for a disorder
associated with elevated levels of ROR1. Generally, the method
includes administering a therapeutically effective amount of an
isolated antibody of the invention to the subject. The antibody can
be any anti-ROR1 antibody of the invention as described herein.
Thus, the antibody can be chimeric, humanized, synthetic, F(ab)2,
Fv, scFv, IgG.DELTA.CH2, F(ab')2, scFv2CH3, Fab, VL, VH, scFv4,
scFv3, scFv2, dsFv, Fv, or (scFv)2. In some embodiments, the method
includes administering an IgG, an scFv, a dsFv, a F(ab').sub.2, a
diabody, or a bivalent antibody. The administered antibody can be
conjugated to a synthetic molecule described above, e.g., a
cytotoxic, cytostatic, or antiangiogenic agent, a therapeutic
radioisotope, or a liposome. An exemplary cytotoxic agent is
Pseudomonas exotoxin A (PE38). Disorders that can be treated
include, for example, B-CLL and MCL. Other disorders associated
with elevated ROR1 that can be treated include Burkitt lymphoma,
renal cell carcinoma, colon cancer (e.g., colon adenocarcinoma),
and breast cancer (e.g., breast adenocarcinoma).
[0062] The invention also provides a method of treating a subject
that has, is suspected to have, or is at risk for a disorder
associated with elevated levels of ROR1 by adoptive transfer of the
genetically engineered T-cells described herein, which express an
antibody of the invention as a T-body (or CAR) that selectively
binds ROR1. Recombinant technology can be used to introduce T-body
(or CAR) encoding genetic material into any suitable T-cells, e.g.,
central memory T-cells from the subject to be treated. The T-cells
carrying the genetic material can be expanded (e.g., in the
presence of cytokines). The genetically engineered T-cells are
transferred, typically by infusion, to the patient. The transferred
T-cells of the invention can then mount an immune response against
ROR1 expressing cells in the subject. The adoptive transfer method
can be used, for example, to treat subjects that have or are
suspected to have B-CLL, MCL, Burkitt lymphoma, renal cell
carcinoma, colon cancer (e.g., colon adenocarcinoma), or breast
cancer (e.g., breast adenocarcinoma).
[0063] In some embodiments, the foregoing methods of treatment can
further include co-administering a second therapeutic agent for the
disorder associated with elevated ROR1. For example, when the
disorder to be treated involves an ROR1-expressing cancer, the
method can further include co-administration of a cytotoxic,
cystostatic, or antiangiogenic agent suitable for treating the
cancer. If the cancer is a B-cell malignancy, the method can
further include, for example, co-administration of rituximab,
alemtuzumab, ofatumumab, or a CHOP chemotherapeutic regimen.
[0064] The terms "treat," "treating," "treatment," and
"therapeutically effective" used herein do not necessarily imply
100% or complete treatment. Rather, there are varying degrees of
treatment recognized by one of ordinary skill in the art as having
a potential benefit or therapeutic effect. In this respect, the
inventive method can provide any amount of any level of treatment.
Furthermore, the treatment provided by the inventive method can
include the treatment of one or more conditions or symptoms of the
disease being treated.
[0065] In another embodiment, the invention provides a method of
detecting in a test sample an altered level of ROR1 (e.g., cell
surface ROR1), for example, relative to a control. Generally, the
method includes contacting a test sample with an antibody of the
invention and determining the amount of antibody that selectively
binds to material (e.g., cells) in the sample to thereby determine
the level of ROR1 in the test sample. A test sample can be from a
cell culture or from a test subject, e.g., a plasma or a tissue
sample from a subject that has, is suspected to have, or is at risk
for a disease or condition associated with elevated ROR1 in a
subject. A control level desirably corresponds to the ROR1 level
detected using the same antibody in a corresponding sample(s) from
one or more control cultures or subjects. Methods of using the
antibody of the invention to determine ROR1 levels can include any
immunoassay such as immuno- (Westem) blotting, enzyme-linked
immunosorbent assay (ELISA), and flow cytometry, e.g.,
fluorescence-activated cell sorting (FACS) analysis.
[0066] The method of detection can be used to screen for the
presence of a disorder associated with elevated ROR1. The method
includes obtaining a sample from a test subject in need of
screening, e.g., a subject that has, is suspected to have, or is at
risk for a disorder associated with elevated ROR1. The level of
ROR1 (e.g., the amount or concentration) in the sample is measured
using an antibody of the invention, and the level in the sample is
compared to a control level of ROR1. The control level represents,
for example, the mean level (e.g., the amount or concentration) in
sample(s) from one or, preferably, multiple control group subjects
that do not have a disorder associated with elevated ROR1.
Alternatively, the control level can correspond to the level or
mean level of ROR1 in one or more samples taken from the test
subject at one or more prior times, such as when the test subject
did not have or did not exhibit, a condition associated with
elevated ROR1. A significantly higher level of ROR1 in the test
sample relative to the control level is indicative of a disorder
associated with elevated ROR1 in the subject.
[0067] In subjects such as humans, where cell surface ROR1
expression is largely restricted to embryonic development, a
control level of ROR1 can be zero or none. Thus, in some
embodiments of the method of the detection provided by the
invention, any significant and detectable amount of ROR1 in a test
sample can be indicative of a disorder associated with elevated
ROR1 in the subject.
[0068] Additionally, the method of detection can be used to monitor
the progress of a disorder associated with elevated ROR1. The
method includes obtaining a sample from a subject in need of
screening, e.g., a subject having been diagnosed or suspected to
have a disorder associated with elevated ROR1. The level of ROR1 in
the sample is measured using an antibody of the invention, and the
level in the sample is compared to a control level corresponding to
the level or mean level of ROR1 in one or more samples taken from
the test subject at one or more prior times. Levels of ROR1 that
are significantly elevated or decreased relative to control
indicate that the subject's disorder is deteriorating or improving,
respectively.
[0069] The foregoing method of detection can be used to screen for
the presence or to monitor the progress of disorders including, for
example, B-CLL, MCL, Burkitt lymphoma, renal cell carcinoma, colon
cancer (e.g., colon adenocarcinoma), and breast cancer (e.g.,
breast adenocarcinoma).
[0070] The invention provides a method for screening a subject for
an altered level of ROR1. Generally, the method includes
administering to the subject an antibody of the invention that is
conjugated to a label (e.g., a contrast agent), imaging the subject
in a manner suitable for detecting the label, and determining
whether a region in the subject has an altered density or
concentration of label as compared to the background level of label
in proximal tissue. Alternatively, the method includes determining
whether there is an altered density or concentration of label in a
region as compared to the density or concentration of label
previously detected in the same region of the subject. Methods of
imaging a subject can include x-ray imaging, x-ray computed
tomography (CT) imaging (e.g., CT angiography (CTA) imaging),
magnetic resonance (MR) imaging, magnetic resonance angiography
(MRA), nuclear medicine, ultrasound (US) imaging, optical imaging,
elastography, infrared imaging, microwave imaging, and the like, as
appropriate for detecting the label conjugated to the antibody. In
a preferred embodiment, the subject has, is suspected to have, or
is at risk for an ROR1-expressing tumor, such as B-CLL, MCL,
Burkitt lymphoma, renal cell carcinoma, tumor of the colon (e.g.,
colon adenocarcinoma), or breast tumor (e.g., breast
adenocarcinoma), and the method is used to screen for or detect the
presence of the tumor. In another embodiment, the method can be
used to monitor the size or density of an ROR1-expressing tumor
over time, e.g., during a course of treatment.
[0071] The invention also provides a pharmaceutical composition
comprising an antibody as described above and a pharmaceutically
acceptable carrier. Pharmaceutical compositions can be prepared
from any of the antibodies described herein. Exemplary compositions
include one or more of a chimeric antibody having SEQ ID NO: 1
(light chain) and/or SEQ ID NO: 2 (heavy chain), a chimeric
antibody having SEQ ID NO: 3 (light chain) and/or SEQ ID NO: 4
(heavy chain), and a chimeric antibody having SEQ ID NO: 5 (light
chain) and/or SEQ ID NO: 6 (heavy chain). Another exemplary
composition comprises a humanized antibody having one, two, three,
four, five, or six CDRs selected from the group consisting of SEQ
ID NOs: 31-48. In preferred embodiments, however, the antibody
includes three CDR sequences of the same light or heavy chain,
e.g., SEQ ID NOs: 31-33, SEQ ID NOs: 34-36; SEQ ID NOs: 37-39; SEQ
ID NOs: 40-42; SEQ ID NOs: 43-45; or SEQ ID NOs: 46-48. In more
preferred embodiments, the composition includes an antibody having
six CDR sequences of the same antibody, e.g., (a) SEQ ID NOs: 31-33
and SEQ ID NOs: 40-42; (b) SEQ ID NOs: 34-36 and SEQ ID NOs: 43-45;
or (c) SEQ ID NOs: 37-39 and SEQ ID NOs: 46-48. Still another
exemplary pharmaceutical composition includes a dsFv fragment,
which can include one or more modifications to the amino acid
sequence as appropriate and understood by one of ordinary skill in
the art.
[0072] The composition of the invention comprises a carrier for the
antibody, desirably a pharmaceutically acceptable carrier. The
pharmaceutically acceptable carrier can be any suitable
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier" as used herein means one or more compatible
solid or liquid fillers, diluents, other excipients, or
encapsulating substances which are suitable for administration into
a human or veterinary patient (e.g., a physiologically acceptable
carrier or a pharmacologically acceptable carrier). The term
"carrier" denotes an organic or inorganic ingredient, natural or
synthetic, with which the active ingredient is combined to
facilitate the use of the active ingredient, e.g., the
administration of the active ingredient to a subject. The
pharmaceutically acceptable carrier can be co-mingled with one or
more of the active components, e.g., a hybrid molecule, and with
each other, when more than one pharmaceutically acceptable carrier
is present in the composition, in a manner so as not to
substantially impair the desired pharmaceutical efficacy.
"Pharmaceutically acceptable" materials typically are capable of
administration to a subject, e.g., a patient, without the
production of significant undesirable physiological effects such as
nausea, dizziness, rash, or gastric upset. It is, for example,
desirable for a composition comprising a pharmaceutically
acceptable carrier not to be immunogenic when administered to a
human patient for therapeutic purposes.
[0073] The pharmaceutical composition can contain suitable
buffering agents, including, for example, acetic acid in a salt,
citric acid in a salt, boric acid in a salt, and phosphoric acid in
a salt. The pharmaceutical composition also optionally can contain
suitable preservatives, such as benzalkonium chloride,
chlorobutanol, parabens, and thimerosal.
[0074] The pharmaceutical composition can be presented in unit
dosage form and can be prepared by any suitable method, many of
which are well known in the art of pharmacy. Such methods include
the step of bringing the antibody of the invention into association
with a carrier that constitutes one or more accessory ingredients.
In general, the composition is prepared by uniformly and intimately
bringing the active agent into association with a liquid carrier, a
finely divided solid carrier, or both, and then, if necessary,
shaping the product.
[0075] A composition suitable for parenteral administration
conveniently comprises a sterile aqueous preparation of the
inventive composition, which preferably is isotonic with the blood
of the recipient. This aqueous preparation can be formulated
according to known methods using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
also can be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example,
as a solution in 1,3-butane diol. Among the acceptable vehicles and
solvents that can be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil can be employed, such as synthetic
mono-or di-glycerides. In addition, fatty acids such as oleic acid
can be used in the preparation of injectables. Carrier formulations
suitable for oral, subcutaneous, intravenous, intramuscular, etc.
administrations can be found in Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton, Pa.
[0076] The delivery systems useful in the context of the invention
include time-released, delayed release, and sustained release
delivery systems such that the delivery of the inventive
composition occurs prior to, and with sufficient time to cause,
sensitization of the site to be treated. The inventive composition
can be used in conjunction with other therapeutic agents or
therapies. Such systems can avoid repeated administrations of the
inventive composition, thereby increasing convenience to the
subject and the physician, and may be particularly suitable for
certain compositions of the invention.
[0077] Many types of release delivery systems are available and
known to those of ordinary skill in the art. Suitable release
delivery systems include polymer base systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are lipids
including sterols such as cholesterol, cholesterol esters, and
fatty acids or neutral fats such as mono-di-and tri-glycerides;
hydrogel release systems; sylastic systems; peptide based systems;
wax coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the active composition is contained in a form within a matrix
such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,
4,748,034, and 5,239,660 and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In
addition, pump-based hardware delivery systems can be used, some of
which are adapted for implantation.
[0078] The term "subject" is used herein, for example, in
connection with therapeutic and diagnostic methods, to refer to
human or animal subjects. Animal subjects include, but are not
limited to, animal models, such as, mammalian models of conditions
or disorders associated with elevated ROR1 expression such as
B-CLL, MCL, Burkitt lymphoma, renal cell carcinoma, colon cancer,
(e.g., colon adenocarcinoma), and breast cancer (e.g., breast
adenocarcinoma).
[0079] The invention also provides kits suitable for carrying out
the methods of the invention. Typically, a kit comprises two or
more components required for performing a therapeutic or detection
method of the invention. Kit components include, but are not
limited to, one or more antibodies of the invention, appropriate
reagents, and/or equipment.
[0080] A kit can comprise an antibody of the invention and an
immunoassay buffer suitable for detecting ROR1 (e.g. by ELISA, flow
cytometry, magnetic sorting, or FACS). The kit may also contain one
or more microtiter plates, standards, assay diluents, wash buffers,
adhesive plate covers, magnetic beads, magnets, and/or instructions
for carrying out a method of the invention using the kit. The kit
can include an antibody of the invention bound to a substrate
(e.g., a multi-well plate or a chip), which is suitably packaged
and useful to detect ROR1. In some embodiments, the kit includes an
antibody of the invention that is conjugated to a label, such as, a
fluorescent label, a biologically active enzyme label, a
luminescent label, or a chromophore label. The kit can further
include reagents for visualizing the conjugated antibody, e.g., a
substrate for the enzyme. In some embodiments, the kit includes an
antibody of the invention that is conjugated to a contrast agent
and, optionally, one or more reagents or pieces of equipment useful
for imaging the antibody in a subject.
[0081] Generally the antibody of the invention in a kit is suitably
packaged, e.g., in a vial, pouch, ampoule, and/or any container
appropriate for a therapeutic or detection method. Kit components
can be provided as concentrates (including lyophilized
compositions), which may be further diluted prior to use, or they
can be provided at the concentration of use. For use of the
antibody of the invention in vivo, single dosages may be provided
in sterilized containers having the desired amount and
concentration of components.
[0082] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0083] This example demonstrates the preparation of monoclonal Fab
antibodies with specificity for ROR1.
[0084] The three extracellular domains of human ROR1 were expressed
alone (hROR1ECD) or as a fusion protein with the Fc domain of human
IgG1 (Fc-hROR1) (FIG. 1). Purified Fc-hROR1 and hROR1ECD were used
to immunize and boost two groups of rabbits of the b9 allotype to
prepare chimeric rabbit/human Fab libraries as described in Popkov,
J. Mol. Biol., 325(2): 325-335 (2003). A total of six b9 allotype
rabbits were used. Four rabbits were immunized and boosted three
times with 100 .mu.g Fc-hROR1, using Freund's complete and
incomplete adjuvant (Sigma-Aldrich; St. Louis, Mo.) for two rabbits
and TiterMax adjuvant (Sigma-Aldrich) for the other two rabbits.
Library R was based on these four rabbits. Library Y was based on
two additional rabbits that were immunized with 100 .mu.g Fc-hROR1
and boosted three times with 100 .mu.g hRORECD using Ribi
(Sigma-Aldrich) adjuvant. Spleen and bone marrow from both femurs
of each rabbit were collected five days after the final boost and
processed for total RNA preparation and RT-PCR amplification of
rabbit V.sub..kappa., V.sub..lamda., and V.sub.H encoding sequences
using established primer combinations and protocols as described in
Rader, Methods Mol. Biol., 525: 101-128, xiv (2009). Rabbit
VL/human C/rabbit VH segments were assembled in one fusion step
based on 3-fragment overlap extension PCR, digested with SfiI, and
cloned into pC3C. Transformation of E. coli strain XL1-Blue
(Stratagene; La Jolla, Calif.) by electroporation yielded
approximately 2.5.times.10.sup.8 and 1.4.times.10.sup.8 independent
transformants for libraries R and Y, respectively.
[0085] Using VCSM13 helper phage (Stratagene), the phagemid
libraries were converted to phage libraries and selected by panning
against immobilized protein. Libraries R and Y were selected in
parallel by four rounds of panning against hROR1ECD. In addition,
library Y was selected by three rounds of panning on hROR1ECD
followed by a final panning round on Fc-hROR1. During the panning
against immobilized Fc-hROR1, unspecific polyclonal human IgG
antibodies (Thermo Scientific; Rockford, Ill.) were added as decoy
at a final concentration of 1 .mu.g/.mu.L. Supernatants of
IPTG-induced selected clones were analyzed by ELISA using
immobilized hROR1ECD and Fc-hROR1 and by flow cytometry using HEK
293F cells stably transfected with human ROR1 (Kwong et al., J.
Mol. Biol., 384(5): 1143-56 (2008)). Rat anti-HA mAb 3F10
conjugated to horse radish peroxidase (Roche) was used in ELISA at
a concentration of 50 ng/mL. The absorbance was measured at 405 nm
using a VersaMax microplate reader (Molecular Devices; Sunnyvale,
Calif.) and SoftMax Pro software (Molecular Devices). Rat anti-HA
mAb 3F10 conjugated to biotin was used in flow cytometry at a
concentration of 5 .mu.g/mL. Fluoresence intensity was analyzed
using a FACSCalibur instrument (BD Biosciences) and FlowJo
analytical software (TreeStar, Ashland, Oreg.).
[0086] Repeated clones were identified by DNA fingerprinting with
AluI, and the V.sub.L and V.sub.H sequences of unique clones were
determined by DNA sequencing as described in Rader, Methods Mol.
Biol., 525: 101-128, xiv (2009).
[0087] As summarized in Table 1, seven different chimeric
rabbit/human Fab clones that bound to hROR1ECD were identified.
TABLE-US-00001 TABLE 1 Panel of chimeric rabbit/human Fab selected
by phage display. Panning rounds Binding hROR1 Fc- hROR1 Fc- HEK
Clone.sup.1 Library ECD hROR1 Repeats ECD.sup.2 hROR1.sup.2
293F/hROR1.sup.3 R11 R 4 0 26/31 ++ ++ + R12 R 4 0 1/31 ++ ++ ++ Y4
Y 4 0 2/31 ++ - - Y13 Y 4 0 14/31 ++ - - Y14 Y 4 0 2/31 + - - Y27 Y
4 0 13/31 ++ - - Y31 Y 3 1 4/4 + + + .sup.1Defined by unique DNA
fingerprint and sequence. .sup.2As measured by ELISA. .sup.3As
measured by flow cytometry.
[0088] Of the seven clones provided in Table 1, three clones
(designated R11, R12, and Y31) also bound to Fc-hROR1 and cell
surface human ROR1 expressed by stably transfected HEK 293F cells
as described in Kwong et al., J. Mol. Biol., 384(5): 1143-56
(2008). The expression cassettes encoding Fab R11, R12, and Y31
were transferred by SfiI cloning into a Fab-(His).sub.6 expression
cassette in vector pET11a with an IPTG-inducible T7 promoter (Stahl
et al., J. Mol. Biol., 397(3): 697-708 (2010)) to remove the HA tag
and gene III fragment encoding sequences of pC3C (Hofer et al., J.
Immunol. Methods, 318(1-2): 75-87 (2007)), and to add a C-terminal
(His).sub.6 tag. Following transformation into E. coli strain
BL21-CodonPlus(DE3)-RIL (Stratagene) and expression through IPTG
induction, Fab R11, R12, and Y31 were purified from bacterial
supernatants by Immobilized Metal Ion Affinity Chromatography using
a 1-mL HisTrap column (GE Healthcare) as described in Kwong, K. Y.
and C. Rader, Curr. Protoc. Protein Sci., Chapter 6: Unit 6, 10
(2009), followed by gel filtration chromatography using a Superdex
200 10/300 GL column with an AKTA FPLC instrument (GE Healthcare).
The quality and quantity of purified Fab was analyzed by SDS-PAGE
and absorbance at 280 nm, respectively, and the variable domains of
R11, R12, and Y31 were sequenced.
[0089] As depicted in FIG. 2, the diverse amino acid sequences of
both frameworks and complementarity determining regions of the
rabbit variable domains of R11, R12, and Y31 revealed unrelated
V.sub..kappa. (R11, Y31), V.sub..lamda. (R12), and V.sub.H
germlines.
[0090] These results demonstrate the production of Fab antibodies
to ROR1.
Example 2
[0091] This example demonstrates the preparation of monoclonal IgG
antibodies with specificity for ROR1.
[0092] For the expression of R11, R12, and Y31 in IgG1 format,
vector PIGG was used as described in Popkov et al., J. Mol. Biol.,
325(2): 325-335 (2003). In this vector, .gamma.1 heavy and .kappa.
light chains are expressed by an engineered bidirectional CMV
promoter cassette. The V.sub.H encoding sequences of Fab R11 and
R12 were PCR amplified using primers
R11-VH-5'(gaggaggagctcactcccagtcggtgaaggagtccga [SEQ ID NO: 49])
and P14-VH-5'(Hofer et al., J. Immunol. Methods, 318(1-2): 75-87
(2007)), respectively, in combination with
R11-12-VH-3'(ccgatgggcccttggtggaggctgaggagatggtgaccagggtgcctggtccccagatg
[SEQ ID NO: 50]), and cloned via ApaI/SacI into PIGG. The light
chain encoding sequences of Fab R11 and R12 were PCR amplified
using primers P14-light-5' (Hofer et al., J. Immunol. Methods,
318(1-2): 75-87 (2007)) and R12-light-5'
(gaggagaagcttgttgctctggatctctggtgcctacggggaactcgtgctgactcagtc [SEQ
ID NO: 51]), respectively, in combination with primer C-kappa-3'
(Hofer et al., J. Immunol. Methods, 318(1-2): 75-87 (2007)), and
cloned via HindIII/XbaI into PIGG with the corresponding heavy
chain encoding sequence.
[0093] The resulting chimeric rabbit/human light chain of R12 is
composed of a rabbit V.sub..lamda. and a human C.sub..kappa.
domain. The V.sub.H encoding sequence of Fab Y31 was PCR amplified
using primers M5-VH-5' and M5-VH-3' (Hofer et al., J. Immunol.
Methods, 318(1-2): 75-87 (2007)), and cloned via ApaI/SacI-ligation
into PIGG. To remove an internal HindIII site by silent mutation,
two fragments of the light chain encoding sequence of Fab Y31 were
PCR amplified using primers P14-light-5' in combination with
Y31-light-3'(attggatgcataatagatcagtagcttgggaggctg [SEQ ID NO: 52])
and Y31-light-5'(aaccagggcagcctcccaagctactgatct [SEQ ID NO: 53]) in
combination with C-kappa-3', fused by overlap extension PCR using
primers P14-light-5' and C-kappa-3', and cloned via HindIII/XbaI
into PIGG with the corresponding heavy chain encoding sequence. The
resulting PIGG-R11, PIGG-R12, and PIGG-Y31 plasmids were
transiently transfected into human HEK 293F cells (Invitrogen;
Carlsbad, Calif.) with 293fectin (Invitrogen), and purified by 1-mL
recombinant Protein A HiTrap column (GE Healthcare, Piscataway,
N.J.) as described in Hofer et al., J. Immunol. Methods, 318(1-2):
75-87 (2007). The quality and quantity of purified IgG1 was
analyzed by SDS-PAGE and A.sub.280 absorbance, respectively. These
results demonstrate the production of IgG antibodies to ROR1.
Example 3
[0094] This example demonstrates specificity and epitope mapping of
Fab and IgG chimeric rabbit/human antibodies to ROR1.
[0095] R11, R12, and Y31 were prepared as Fab and IgG. Fab regions
were prepared as described in Example 1. IgG chimeric rabbit/human
antibodies were prepared as described in Example 2. The specificity
of the purified Fab and IgG1 was probed by ELISA with an extended
panel of recombinant ROR1 proteins that included Fc-hROR1, its
mouse analogue Fc-mROR1, and five Fc fusion proteins with only one
or two extracellular domains of human ROR1 as shown in FIG. 1. Also
included was commercially available hROR2-Fc (R&D Systems;
Minneapolis, Minn.). Chimeric rabbit/human Fab and IgG1 P14 against
NgR2 (Hofer et al., J. Immunol. Methods, 318(1-2): 75-87 (2007))
was used as negative control. Fab (data not shown) and IgG1 (FIGS.
3A and 3B) revealed identical binding patterns. As shown in FIG.
3A, IgG1 R11, R12, and Y31 bound to human ROR1, but not to human
ROR2. In addition, IgG1 R11 and Y31 were found to be cross-reactive
with mouse ROR1. The binding of IgG1 R11, R12, and Y31 to only one
or two extracellular domains of human ROR1 (FIG. 3B) confirmed the
recognition of three different epitopes. In selectively recognizing
Fc-hROR1kr and Fc-hROR1kr+fz, IgG1 R11 was the only mAb that mapped
to a single domain. In contrast, IgG1 R12 and Y31 selectively
recognized Fc-hROR1ig+fz and Fc-ROR1fz+kr, respectively, but not
any of the single domains, thereby indicating that the epitopes of
these mAbs either are located in the region that links two
neighboring domains, i.e. at the conjunction of Ig and Fz domains
in case of R12 and at the conjunction of Fz and Kr domains in case
of Y31, or bind to conformational epitopes that require the
presence of these two neighboring domains.
[0096] The three epitopes of IgG1 R11, R12, and Y31 were found to
encompass a large portion of the extracellular region of human
ROR1. To investigate the therapeutic implications of membrane
distal and proximal binding of anti-ROR1 mAbs, the independence of
the three epitopes was also analyzed by surface plasmon resonance
using a Biacore X100 (GE Healthcare, Piscataway, N.J.) instrument.
Studies were performed using surface plasmon resonance for the
measurement of the affinities of Fab R11, R12, and Y31 and the
virtual affinities of IgG1 R11, R12, and Y31 to Fc-hROR1 and
Fc-mROR1, as well as for epitope mapping. For affinity
measurements, CM5 sensor chips were activated for immobilization
with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
and N-hydroxysuccinimide. Fc-hROR1 and Fc-mROR1 fusion proteins in
10 mM sodium acetate (pH 5.0) were immobilized at a density of 669
resonance units (RU) for Fc-hROR1 and 429 RU for Fc-mROR1 in two
flow cells on separate sensor chips. Subsequently, the sensor chips
were deactivated with 1M ethanolamine hydrochloride (pH 8.5). Each
sensor chip included an empty flow cell for instantaneous
background depletion. All binding assays used 1.times.HBS-EP+
running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA (pH 7.4), and
0.05% (v/v) Surfactant P20) and a flow rate of 30 .mu.L/min. Fab
and IgG1 R11, R12, and Y31 were injected at five or six different
concentrations ranging from 1.5 to 100 nM in duplicates. The sensor
chips were regenerated with glycine-HCl (pH 2.0) without any loss
of binding capacity. Calculation of association (k.sub.on) and
dissociation (k.sub.off) rate constants was based on a 1:1 Langmuir
binding model. The equilibrium dissociation constant (K.sub.d) was
calculated from k.sub.off/k.sub.on. For epitope mapping studies,
Fc-hROR1 was immobilized on a CM5 sensor chip at a density of 219
RU. IgG1 R11, R12, and Y31 were prepared as 300 nM solution in
1.times.HBS-EP+ running buffer. In the first cycle, IgG1 R11 was
injected first, followed by a mixture of IgG1 R11 and R12, followed
finally by a mixture of IgG1 R11, R12, and Y31. IgG1 R11 or IgG1
R11 in combination with IgG1 R12 were included in these mixtures to
prevent signal loss due to dissociation. In the second cycle the
injection order was R11, R11+Y31, and R11+Y31+R12. Analogously, R12
was injected first in the third and fourth cycle, and Y31 was
injected first in the fifth and sixth cycle. RU increases that
exceeded the values found for IgG1 R11, R12, and Y31 alone
indicated independent epitopes that allow simultaneous binding.
[0097] As shown in FIG. 4, IgG1 R11 and R12 were found to bind
simultaneously and independently to Fc-hROR1 regardless of the
sequence of injection. By contrast, IgG1 R11, but not IgG1 R12, was
found to block the binding of IgG1 Y31 when injected first or
compete with the binding of IgG1 Y31 when injected second. Surface
plasmon resonance also revealed the simultaneous binding of Fab R11
and R12 to Fc-hROR1 (data not shown).
[0098] These results demonstrate that the epitopes of R11 in the Kr
domain and Y31 at the conjunction of Fz and Kr domains partially
overlap, whereas R12 binds to an independent epitope at the
conjunction of Ig and Fz domains.
Example 4
[0099] This example demonstrates various binding properties of mAbs
R11, R12, and Y31 in IgG and Fab format.
[0100] Surface plasmon resonance with the Biacore X100 instrument
(GE Healthcare, Piscataway, N.J.), as described in Example 3, was
used to measure the affinity and avidity of mAbs R11, R12, and Y31
in Fab and IgG1 format, respectively, as shown in Table 2, FIG. 5A,
and FIG. 5B. Fab R12 was found to be the strongest binder with an
affinity of 0.56 nM to Fc-hROR1. Fab R11 and Y31 revealed
affinities of 2.7 and 8.8 nM, respectively. An approximately
twenty-fold slower dissociation rate was determined for Fab R12,
whereas Fab R11 was found to have a faster association rate.
Conversion from monovalent Fab to bivalent IgG1 increased the
virtual affinity of R11, R12, and Y31 by factor 14, 5, and 12,
respectively; all three IgG1 revealed subnanomolar avidity.
Confirming the ELISA data, R11 and Y31 revealed comparable
affinities and avidities for Fc-hROR1 and Fc-mROR1, indicating that
their epitopes are entirely conserved between human and mouse ROR1.
By contrast, R12 did not reveal detectable binding to Fc-mROR1.
TABLE-US-00002 TABLE 2 MAb Antigen k.sub.on (10.sup.5)
(M.sup.-1s.sup.-1) k.sub.off (10.sup.-4) (s.sup.-1) K.sub.d (nM)
Fab R11 Fc-hROR1 20.4 54.7 2.7 Fc-mROR1 16.9 50.4 3.0 IgG1 11
Fc-hROR1 19.4 3.6 "0.19" Fc-mROR1 9.9 3.0 "0.30" Fab R12 Fc-hROR1
5.5 3.1 0.56 Fc-mROR1 no binding no binding no binding IgG1 R12
Fc-hROR1 5.5 0.62 "0.11" Fc-mROR1 no binding no binding no binding
Fab Y31 Fc-hROR1 8.5 75.2 8.8 Fc-mROR1 9.1 38.3 4.2 IgG1 Y31
Fc-hROR1 4.9 3.5 "0.71" Fc-mROR1 5.4 2.4 "0.44"
[0101] As described in Example 1, Fab R11, R12, and Y31 recognized
cell surface human ROR1 expressed by stably transfected HEK 293F
cells. Flow cytometry was used to validate the selective binding of
IgG1 R11, R12, and Y31 to JeKo-1 and HBL-2 cells (FIG. 6A and FIG.
6B). JeKo-1 and HBL-2 are human mantle cell lymphoma cell lines
that express ROR1 at similar levels as primary human CLL cells.
Cells were stained using standard flow cytometry methodology.
Briefly, for anti-ROR1 Fab, cells were stained with unpurified or
purified Fab on ice for 1 h. After washing twice with ice-cold flow
cytometry buffer (PBS containing 1% (v/v) FBS), cells were
incubated with 5 .mu.g/mL of biotinylated rat anti-HA mAb 3F10
(Roche) in flow cytometry buffer on ice for 1 h, washed as before,
and stained with PE-streptavidin (BD Biosciences) on ice for 30
min. For anti-ROR1 IgG1, cells were first blocked with hIgG at room
temperature for 20 min, then incubated on ice for 1 h with
biotinylated anti-ROR1 IgG1 alone (for HEK 293F/hROR1, JeKo-1, and
HBL-2 cells) or in combination with FITC-CD19/APC-CD5 (BD
Biosciences; Franklin Lakes, N.J.) (for PBMC from untreated CLL
patients). After washing twice with ice-cold flow cytometry buffer,
cells were stained with PE-streptavidin on ice for 30 min.
Propidium iodide (PI) was added to a final concentration of 5
.mu.g/mL to exclude dead cells from analysis. Cells were analyzed
using a FACSCalibur instrument (BD Biosciences) and FlowJo
analytical software (TreeStar, Ashland, Oreg.).
[0102] Human anti-tetanus toxoid mAb TT11 in IgG1 format (Kwong et
al., J. Mol. Biol., 384(5): 1143-56 (2008)) was used as a negative
control, as shown in Table 3, which sets forth the data on flow
cytometry binding of IgG1 R11, R12, and Y31 to primary CLL cells
from one representative patient (shown in units of mean
fluorescence intensity (MFI)).
TABLE-US-00003 TABLE 3 0.01 0.1 1 5 10 .mu.g/mL .mu.g/mL .mu.g/mL
.mu.g/mL .mu.g/mL IgG1 R11 Not 6.6 18.1 64.9 137.7 determined IgG1
R12 36.4 89.4 97.9 121.8 Not determined IgG1 Y31 Not 5.4 8.3 21.6
58.9 determined IgG1 TT11 4.9 4.8 7.2 7.3 7.3
[0103] IgG1 R12 demonstrated strong and homogeneous binding at
concentrations as low as 0.01 .mu.g/mL (67 pM), confirming its
subnanomolar avidity found by surface plasmon resonance. By
contrast, the binding of IgG1 R11 and, in particular, Y31 was
somewhat weaker and more heterogeneous. This pattern correlates
with the different avidities found for the three mAbs, and is
supported by the accessibility of the three different epitopes on
cell surface ROR1. The presumed membrane distal epitope of R12 at
the conjunction of Ig and Fz domains improve access for the bulky
IgG1 format as compared with the presumed membrane proximal epitope
of R11 and Y31 in the Kr domain and at the conjunction of Fz and Kr
domains, respectively. In fact, conversion of R11 to the less bulky
scFv-Fc format (.about.100 kDa; two polypeptide chains)
demonstrated significantly stronger binding at lower concentrations
compared to the IgG1 format (.about.150 kDa; four polypeptide
chains) (data not shown).
[0104] The binding of IgG1 R11, R12, and Y31 was analyzed against
PBMC prepared from five untreated CLL patients. Chimeric
rabbit/human IgG1 P14 against NgR2 served as negative control.
Representative flow cytometry plots from one CLL patient as
compared to negative controls are shown in FIG. 6B. Consistent with
the results of Baskar et al., Clin. Cancer Res., 14(2): 396-404
(2008) (goat anti-human ROR1 pAbs), IgG1 R11, R12, and Y31
selectively bound to CLL cells (CD5+CD19+), but not to normal B
cells (CD5-CD19+), T cells (CD5+CD19-), and CD5-CD19- PBMC from
untreated CLL patients. The pattern of binding to primary CLL cells
was similar to that noted for the JeKo-1 cell line, namely strong
and homogeneous binding of IgG1 R12, and weaker and more
heterogeneous binding of IgG1 R11 and Y31. Additional flow
cytometry plots showing the binding of IgG1 R12 to PBMC prepared
from an additional four CLL patients are shown in FIG. 7A, FIG. 7B,
FIG. 7C, and FIG. 7D. Gating for normal NK cells, T cells, and B
cells in these CLL patients further confirmed the specificity of
IgG1 R12 for CLL cells.
[0105] The foregoing results demonstrate that IgG1 R11, R12, and
Y31 have subnanomolar avidity for ROR1 and can be used to
specifically distinguish (i) tumor cells obtained from lymphoma
patients from (ii) normal B-cells taken from healthy subjects.
Example 5
[0106] This example evaluates the complement-dependent cytotoxic
(CDC) properties of chimeric rabbit/human anti-ROR1 antibodies.
[0107] As target cells, JeKo-1 and HBL-2 cells or cryopreserved
PBMC from untreated CLL patients were harvested, washed, and
resuspended in RPMI 1640 containing 10% (v/v) FBS, 100 U/mL
penicillin, and 100 .mu.g/mL streptomycin, and distributed into
96-well U-bottom plates (Corning; Corning, N.Y.) at a density of
1.times.10.sup.5 cells/well. After incubation for 1 h on ice with
20 .mu.g/mL IgG1 R11, R12, Y31, and P14 (negative control), as well
as unspecific polyclonal human IgG (Thermo Scientific) as a further
negative control and rituximab (Genentech; South San Francisco,
Calif.) as a positive control, the cells were harvested, washed
once with PBS to remove unbound antibodies, and incubated with 20%
complement from 3-4-week-old rabbits (Pel-Freez; Rogers, A R) for 2
h at 37.degree. C. in 5% CO.sub.2. After adding PI to a final
concentration of 5 .mu.g/mL, dead cells were detected by PI
accumulation using a FACSCalibur instrument and FlowJo analytical
software.
[0108] Whereas rituximab mediated potent CDC, none of the other
antibodies revealed cytotoxicity above background (FIG. 8A, FIG.
8B, and FIG. 8C), and neither did a mixture of IgG1 R11 and R12 or
rabbit anti-human ROR1 IgG pAbs purified from the serum of our
immunized rabbits (data not shown).
[0109] These findings do not indicate that ROR1 is a suitable
antigen for mediating CDC by mAbs or pAbs in IgG format.
Example 6
[0110] This example evaluates the antibody-dependent cellular
cytotoxicity (ADCC) properties of chimeric rabbit/human anti-ROR1
antibodies.
[0111] ADCC was assayed in a bioluminescent protease release assay
(Glo Cytotoxicity Assay; Promega, Madison, Wis.) using the
manufacturer's protocol with minor modifications. NK cells from
healthy volunteers prepared from apheresis blood were used as
effector cells. JeKo-1 and HBL-2 cells or cryopreserved PBMC from
untreated CLL patients prepared described in Example 5 were used as
target cells and distributed into 96-well U-bottom plates at a
density of 1.times.10.sup.4 cells/well. The target cells were
preincubated for 1 h at 37.degree. C. with serially diluted (from
20 to 0.02 .mu.g/mL) IgG1 R11, R12, Y31, TT11 (negative control),
and rituximab (positive control). Without washing, effector cells
were added (100 .mu.L/well) at an effector-to-target cell ratio of
20:1 or 25:1 and incubated for 24 h at 37.degree. C. in 5%
CO.sub.2. After centrifugation, 50 .mu.L/well of supernatant was
transferred to a 96-well Costar 3610 white tissue culture plate
followed by addition of 25 .mu.L/well CytoTox-Glo cytotoxicity
assay reagent (Promega, Madison, Wis.). After 15 min at room
temperature, luminescence was measured with a Spectra Max M5
microplate reader (Molecular Devices, Sunnyvale, Calif.). The
percentage of specific cytotoxicity was calculated according to the
formula: Percent specific
cytotoxicity=100.times.(EX-E.sub.spon-T.sub.spon)/(T.sub.max-T.sub.spon),
where EX represents the release from experimental wells, E.sub.spon
is the spontaneous release of effector cells alone, T.sub.spon is
the spontaneous release of target cells alone, and T.sub.max is the
maximum release from target cells lysed in 30 .mu.g/mL digitonin.
Data were computed as mean.+-.standard deviation of
triplicates.
[0112] Rituximab-mediated ADCC demonstrated similar potency against
JeKo-1 and HBL-2 cells (FIG. 9A). This ADCC activity was robust
over a concentration range from 0.02 .mu.g/mL to 20 .mu.g/mL (FIG.
9C). By contrast, ADCC activity was detectable for IgG1 R12 only at
or above 5 .mu.g/mL (FIGS. 9A, C), IgG1 R11 and Y31 were not
significantly different from the negative control. Similar results
are shown in FIG. 9B, which provides ADCC results against PBMC of
untreated CLL patients.
[0113] These results show that IgG R12 has weak ADCC activity but
do not indicate ADCC activity for IgG1 R11 or Y31.
Example 7
[0114] This example provides analysis of the role of
internalization or dissociation in the inability of IgG1 R11, R12,
and Y31 to mediate CDC and ADCC.
[0115] Using a 96-well U-bottom plate, 3.times.10.sup.6
cryopreserved PBMC from untreated CLL patients were first blocked
with 100 .mu.g/mL unspecific polyclonal human IgG at room
temperature for 20 min, then stained with 10 .mu.g/mL biotinylated
IgG1 R11 and Y31, or 1 .mu.g/mL biotinylated IgG1 R12 on ice for 1
h. After washing three times with flow cytometry buffer to remove
unbound antibody, the cells were either left on ice or incubated at
37.degree. C. for 15 min, 30 min, 1 h, and 2 h to facilitate
internalization. In addition, the cells were incubated at
37.degree. C. for 2 h in the presence of 10 .mu.M phenylarsine
oxide (Sigma-Aldrich) to inhibit internalization. Subsequently, the
cells were washed once with flow cytometry buffer and incubated
with PE-streptavidin on ice for 30 min. After three final washes
with flow cytometry buffer, the mean fluorescence intensity (MFI)
of the cells was measured using a FACSCalibur instrument and FlowJo
analytical software.
[0116] MFI reduction can be explained by internalization or
dissociation or a combination of both. The percentage of MFI
reduction was calculated for each mAb relative to the unspecific
polyclonal human IgG control (MFI.sub.background) and mAb
maintained on ice (MFI.sub.max) by using the formula
[(MFI.sub.max-MFI.sub.background)-(MFI.sub.experimental-MFI.sub.b-
ackground)]/(MFI.sub.max-MFI.sub.background).times.100.
[0117] Human ROR1 has previously been shown to mediate
internalization of polyclonal goat anti-human ROR1 IgG by a route
that can be completely blocked by endocytosis inhibitor
phenylarsine oxide (Baskar et al., Clin. Cancer Res., 14(2):
396-404 (2008)). MFI reduction was noted for all three IgG1 after 2
h (FIG. 10B). In case of IgG1 R11 and R12, phenylarsine oxide
completely blocked MFI reduction, revealing internalization as the
dominating factor. By contrast, dissociation contributed to the
continuous disappearance of IgG1 Y31 from the cell surface (FIG.
10B).
[0118] IgG1 R12 internalized more slowly than IgG1 R11 with peaks
at 20-25% after 2 h compared to 50-55%.
[0119] These results provide evidence that the more durable
presence of IgG1 R12 at the cell surface contributes to the weak
ADCC activity noted for IgG1 R12 which was not detected for IgG1
R11 and Y31.
Example 8
[0120] This example demonstrates the construction and
characterization of a disulfide stabilized fragment (dsFv) of
chimeric rabbit/human anti-ROR1 antibodies R11, R12, and Y31 fused
to an immunotoxin.
[0121] A dsFv fragment of mAb R11, R12, or Y31 (dsFv) is generated
and fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38)
generally according to methods described in Pastan et al., Methods
Mol. Biol., 248: 503-518 (2004). The original VH and VL coding
sequences of R11, R12, or Y31 (see FIG. 2) are altered as necessary
to prepare a dsFv fragment. The altered VH coding sequence is
subcloned in-frame with a PE38 coding sequence in a pRB98 vector
carrying a chloramphenicol resistance gene (the vector is described
in Kreitman et al., in Drug Targeting, Francis et al., Eds., Vol.
25, pp. 215-226, Humana Press Inc, Totowa, N.J., 2000). Altered VH
and VL chains are separately expressed in E. coli, and the
resulting proteins are harvested and solubilized. The VH and VL are
refolded together to form dsFv-PE38 fusion immunotoxin, which is
purified by ion-exchange and gel filtration chromatography as
described in Pastan et al., supra, 2004.
[0122] The resulting recombinant dsFv-PE38 immunotoxin conjugates
are evaluated by flow cytometry and compared to chimeric
rabbit/human anti-ROR1 antibodies R11, R12, and Y31 for their
ability to bind to the human ROR1-expressing mantle cell lymphoma
cell lines JeKo-1 and HBL-2. JeKo-1 and HBL-2 cell binding by mAbs
R11, R12, and Y31 is detected using a goat anti-mouse IgG
polyclonal antibody (pAb) conjugated to APC (Jackson ImmunoResearch
Laboratories, West Grove, Pa.) at 1:300 dilution. JeKo-1 and HBL-2
cell binding of dsFv-PE38 immunotoxin conjugates is detected using
rabbit anti-Pseudomonas exotoxin A pAb (1:100 dilution)
(Sigma-Aldrich, St. Louis, Mo.) as a secondary antibody and goat
anti-rabbit IgG pAb conjugated to Cy5 (1:300 dilution) (Jackson
ImmunoResearch Laboratories) as a tertiary antibody. The results
are expected to demonstrate that, despite the inherent monovalency
of a recombinant dsFv-PE38 immunotoxin, binding to native cell
surface ROR1 is detectable at low concentrations.
[0123] An analysis of dsFv-PE38 immunotoxin binding to PBMC from
B-CLL patients is expected to show similar results. Additionally,
ELISA experiments are expected to demonstrate that dsFv-PE38
immunotoxin retains binding specificity for the extracellular
domain of human ROR1.
[0124] The foregoing example provides a method of preparing a
recombinant immunotoxin conjugated antibody of the invention, which
is based on mAb R11, R12, or Y31, and which has conserved binding
specificity for ROR1, including native ROR1 expressed on the cell
surface of malignant B-cells.
Example 9
[0125] This example demonstrates cytotoxic properties of dsFv of
chimeric rabbit/human anti-ROR1 antibodies R11, R12, and Y31 fused
to an immunotoxin applied to ROR1 expressing cells.
[0126] JeKo-1 and HBL-2 cells are cultured in Roswell Park Memorial
Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum
and incubated for 48 hours at 37.degree. C. in a 96-well tissue
culture plate with various doses (0-100 .mu.g/mL) of the dsFv-PE38
immunotoxin prepared in Example 8. The cells are subsequently
analyzed by flow cytometry using annexin V and propidium iodide to
stain apoptotic and dead cells, respectively. The percentage of
cells positive for both annexin V and propidium iodide are
evaluated as a function of the concentration of dsFv-PE38. The
cytotoxicity of dsFv-PE38 includes not only cell death (necrosis)
as evidenced by propidium iodide staining, but also extensive
apoptosis, as evidenced by annexin V staining.
[0127] The foregoing example provides a method of evaluating the
ability of a recombinant immunotoxin conjugated antibody of the
invention, which is based on mAb R11, R12, or Y31, to effect
dose-dependent killing of JeKo-1 and HBL-2 cells at low
concentrations.
Example 10
[0128] This example demonstrates the ability of IgG1 R11, R12, and
Y31 to induce or inhibit apoptosis in primary CLL cells from
patients.
[0129] Apoptosis was evaluated in the presence and absence of fetal
bovine serum (FBS). FBS has been shown to enhance spontaneous
apoptosis of primary CLL cells ex vivo (Levesque et al., Leukemia,
15: 1305-1307 (2001)). Using FBS-free medium, apoptosis induction
was analyzed in PBMC from three CLL patients with 80% or more
CD19+CD5+ROR1+ cells following incubation for three days with IgG1
R11, R12, Y31, TT11, and rituximab alone or in the presence of a
cross-linking pAb. PBMC from CLL patients were distributed into
48-well flat-bottom plates at a density of 5.times.10.sup.5
cells/well in either (i) serum-free AIM-V medium (Invitrogen)
supplemented with 50 .mu.M .beta.-mercaptoethanol (Sigma-Aldrich)
or (ii) RPMI 1640 supplemented with 10% (v/v) heat-inactivated
fetal bovine serum, 100 U/mL penicillin, and 100 .mu.g/mL
streptomycin in the presence or absence of 100 ng/mL recombinant
human IL-4 (R&D Systems) and 1 .mu.g/mL soluble recombinant
human CD40L trimer (Amgen, Thousand Oaks, Calif.). Cells were
incubated with 5 .mu.g/mL IgG1 R11, R12, Y31, TT11, or rituximab at
37.degree. C. in 5% CO.sub.2. For cross-linking, 20 .mu.g/mL
F(ab').sub.2 goat anti-human IgG (Fc-specific, Jackson
ImmunoResearch Laboratories) was added to the cell suspension
simultaneously with primary antibodies. Apoptosis and cell death
was measured by flow cytometry following staining with Alexa Fluor
647 Annexin V (Invitrogen) and SYTOX Green nucleic acid stain
(Invitrogen). Briefly, cells were gently harvested after 72 h
incubation with indicated treatments, washed once with cold
apoptosis binding buffer (140 mM NaCl, 2.5 mM CaCl.sub.2, 10 mM
HEPES, pH 7.4), and resuspended in 200 .mu.L apoptosis binding
buffer. After adding 1 .mu.L Alexa Fluor 647 Annexin V and 1 .mu.L
SYTOX Green to a final concentration of 50 nM, the cells were
incubated for 15 min in the dark at room temperature, resuspended
in 400 .mu.L apoptosis binding buffer, and analyzed using a
FACSCalibur instrument and FlowJo analytical software.
[0130] As shown in FIG. 11A, the only increase in spontaneous
apoptosis was noted for cross-linked rituximab. This was consistent
and reproducible for all three tested PBMC samples. In the presence
of FBS, apoptosis approached 50% after three days (FIG. 11B). As
observed previously, the addition of IL-4 and CD40L strongly
suppressed apoptosis. See, e.g., Baskar et al., Clin. Cancer Res.,
14: 396-404 (2008). IgG1 R11, R12, Y31, and TT11 (negative control)
neither increased nor decreased apoptosis alone or after
cross-linking. They also did not influence the suppression of
apoptosis by IL-4 and CD40L. By contrast, cross-linked rituximab
was found to increase apoptosis and partially override its
suppression (FIG. 11B).
[0131] The induction of apoptosis in MCL cell line HBL-2 also was
investigated (data not shown). In contrast to primary CLL cells,
rituximab alone was sufficient to induce apoptosis in HBL-2 cells.
This activity was further increased after cross-linking.
Nonetheless, IgG1 R11, R12, and Y31 did not induce apoptosis in
HBL-2 cells with or without cross-linking.
[0132] These results demonstrate that this panel of chimeric
rabbit/human IgG1 antibodies neither induces nor inhibits apoptosis
of primary CLL cells.
[0133] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0134] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0135] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
531110PRTOryctolagus cuniculus 1Glu Leu Val Met Thr Gln Thr Pro Ser
Ser Thr Ser Gly Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys
Gln Ala Ser Gln Ser Ile Asp Ser Asn 20 25 30 Leu Ala Trp Phe Gln
Gln Lys Pro Gly Gln Pro Pro Thr Leu Leu Ile 35 40 45 Tyr Arg Ala
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Gln Arg 65 70
75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Val Gly Asn Val
Ser 85 90 95 Tyr Arg Thr Ser Phe Gly Gly Gly Thr Glu Val Val Val
Lys 100 105 110 2116PRTOryctolagus cuniculus 2Gln Ser Val Lys Glu
Ser Glu Gly Asp Leu Val Thr Pro Ala Gly Asn 1 5 10 15 Leu Thr Leu
Thr Cys Thr Ala Ser Gly Ser Asp Ile Asn Asp Tyr Pro 20 25 30 Ile
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40
45 Phe Ile Asn Ser Gly Gly Ser Thr Trp Tyr Ala Ser Trp Val Lys Gly
50 55 60 Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu Lys
Met Thr 65 70 75 80 Ser Leu Thr Thr Asp Asp Thr Ala Thr Tyr Phe Cys
Ala Arg Gly Tyr 85 90 95 Ser Thr Tyr Tyr Cys Asp Phe Asn Ile Trp
Gly Pro Gly Thr Leu Val 100 105 110 Thr Ile Ser Ser 115
3112PRTOryctolagus cuniculus 3Glu Leu Val Leu Thr Gln Ser Pro Ser
Val Ser Ala Ala Leu Gly Ser 1 5 10 15 Pro Ala Lys Ile Thr Cys Thr
Leu Ser Ser Ala His Lys Thr Asp Thr 20 25 30 Ile Asp Trp Tyr Gln
Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met 35 40 45 Gln Val Gln
Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp 50 55 60 Arg
Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro 65 70
75 80 Ser Val Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp
Tyr 85 90 95 Ile Gly Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr
Val Thr Gly 100 105 110 4121PRTOryctolagus cuniculus 4Gln Glu Gln
Leu Val Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly 1 5 10 15 Ser
Leu Thr Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Ser Ala Tyr 20 25
30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45 Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr Tyr Tyr Ala Thr
Trp Val 50 55 60 Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Gln
Asn Thr Val Asp 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
Arg Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Asp Ser Tyr Ala Asp Asp
Gly Ala Leu Phe Asn Ile Trp Gly 100 105 110 Pro Gly Thr Leu Val Thr
Ile Ser Ser 115 120 5108PRTOryctolagus cuniculus 5Glu Leu Val Met
Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr
Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Ile Gly Ser Tyr 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly
Val Gln Arg 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Ser
Leu Ser Asn Ser Asp 85 90 95 Asn Val Phe Gly Gly Gly Thr Glu Leu
Glu Ile Leu 100 105 6117PRTOryctolagus cuniculus 6Gln Ser Leu Glu
Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr
Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Asn Ser His Trp 20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35
40 45 Ile Ile Ala Ala Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys
Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
Arg Ile Ala 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe
Cys Ala Arg Asp Tyr 85 90 95 Gly Asp Tyr Arg Leu Val Thr Phe Asn
Ile Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
723PRTArtificial SequenceSynthetic 7Glu Leu Val Met Thr Gln Thr Pro
Ser Ser Thr Ser Gly Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn
Cys 20 815PRTArtificial SequenceSynthetic 8Trp Phe Gln Gln Lys Pro
Gly Gln Pro Pro Thr Leu Leu Ile Tyr 1 5 10 15 932PRTArtificial
SequenceSynthetic 9Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly
Thr Glu Tyr Thr 1 5 10 15 Leu Thr Ile Ser Gly Val Gln Arg Glu Asp
Ala Ala Thr Tyr Tyr Cys 20 25 30 1010PRTArtificial
SequenceSynthetic 10Phe Gly Gly Gly Thr Glu Val Val Val Lys 1 5 10
1122PRTArtificial SequenceSynthetic 11Glu Leu Val Leu Thr Gln Ser
Pro Ser Val Ser Ala Ala Leu Gly Ser 1 5 10 15 Pro Ala Lys Ile Thr
Cys 20 1219PRTArtificial SequenceSynthetic 12Trp Tyr Gln Gln Leu
Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val 1 5 10 15 Gln Ser Asp
1332PRTArtificial SequenceSynthetic 13Gly Val Pro Asp Arg Phe Ser
Gly Ser Ser Ser Gly Ala Asp Arg Tyr 1 5 10 15 Leu Ile Ile Pro Ser
Val Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys 20 25 30
1411PRTArtificial SequenceSynthetic 14Phe Gly Gly Gly Thr Gln Leu
Thr Val Thr Gly 1 5 10 1523PRTArtificial SequenceSynthetic 15Glu
Leu Val Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10
15 Gly Thr Val Thr Ile Asn Cys 20 1615PRTArtificial
SequenceSynthetic 16Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
Leu Ile Tyr 1 5 10 15 1732PRTArtificial SequenceSynthetic 17Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Thr 1 5 10 15
Leu Thr Ile Ser Gly Val Gln Arg Glu Asp Ala Ala Thr Tyr Tyr Cys 20
25 30 1810PRTArtificial SequenceSynthetic 18Phe Gly Gly Gly Thr Glu
Leu Glu Ile Leu 1 5 10 1929PRTArtificial SequenceSynthetic 19Gln
Ser Val Lys Glu Ser Glu Gly Asp Leu Val Thr Pro Ala Gly Asn 1 5 10
15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Ser Asp Ile Asn 20 25
2014PRTArtificial SequenceSynthetic 20Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile Gly 1 5 10 2130PRTArtificial
SequenceSynthetic 21Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp
Leu Lys Met Thr 1 5 10 15 Ser Leu Thr Thr Asp Asp Thr Ala Thr Tyr
Phe Cys Ala Arg 20 25 30 2211PRTArtificial SequenceSynthetic 22Trp
Gly Pro Gly Thr Leu Val Thr Ile Ser Ser 1 5 10 2330PRTArtificial
SequenceSynthetic 23Gln Glu Gln Leu Val Glu Ser Gly Gly Arg Leu Val
Thr Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
Phe Asp Phe Ser 20 25 30 2414PRTArtificial SequenceSynthetic 24Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ala 1 5 10
2532PRTArtificial SequenceSynthetic 25Arg Phe Thr Ile Ser Ser Asp
Asn Ala Gln Asn Thr Val Asp Leu Gln 1 5 10 15 Met Asn Ser Leu Thr
Ala Ala Asp Arg Ala Thr Tyr Phe Cys Ala Arg 20 25 30
2611PRTArtificial SequenceSynthetic 26Trp Gly Pro Gly Thr Leu Val
Thr Ile Ser Ser 1 5 10 2729PRTArtificial SequenceSynthetic 27Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10
15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Asn 20 25
2814PRTArtificial SequenceSynthetic 28Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile Gly 1 5 10 2930PRTArtificial
SequenceSynthetic 29Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp
Leu Arg Ile Ala 1 5 10 15 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr
Phe Cys Ala Arg 20 25 30 3011PRTArtificial SequenceSynthetic 30Trp
Gly Pro Gly Thr Leu Val Thr Val Ser Ser 1 5 10 3111PRTArtificial
SequenceSynthetic 31Gln Ala Ser Gln Ser Ile Asp Ser Asn Leu Ala 1 5
10 327PRTArtificial SequenceSynthetic 32Arg Ala Ser Asn Leu Ala Ser
1 5 3312PRTArtificial SequenceSynthetic 33Leu Gly Gly Val Gly Asn
Val Ser Tyr Arg Thr Ser 1 5 10 3412PRTArtificial SequenceSynthetic
34Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp 1 5 10
357PRTArtificial SequenceSynthetic 35Gly Ser Tyr Thr Lys Arg Pro 1
5 369PRTArtificial SequenceSynthetic 36Gly Ala Asp Tyr Ile Gly Gly
Tyr Val 1 5 3711PRTArtificial SequenceSynthetic 37Gln Ala Ser Gln
Ser Ile Gly Ser Tyr Leu Ala 1 5 10 387PRTArtificial
SequenceSynthetic 38Tyr Ala Ser Asn Leu Ala Ser 1 5
3910PRTArtificial SequenceSynthetic 39Leu Gly Ser Leu Ser Asn Ser
Asp Asn Val 1 5 10 405PRTArtificial SequenceSynthetic 40Asp Tyr Pro
Ile Ser 1 5 4116PRTArtificial SequenceSynthetic 41Phe Ile Asn Ser
Gly Gly Ser Thr Trp Tyr Ala Ser Trp Val Lys Gly 1 5 10 15
4211PRTArtificial SequenceSynthetic 42Gly Tyr Ser Thr Tyr Tyr Cys
Asp Phe Asn Ile 1 5 10 435PRTArtificial SequenceSynthetic 43Ala Tyr
Tyr Met Ser 1 5 4417PRTArtificial SequenceSynthetic 44Thr Ile Tyr
Pro Ser Ser Gly Lys Thr Tyr Tyr Ala Thr Trp Val Asn 1 5 10 15 Gly
4512PRTArtificial SequenceSynthetic 45Asp Ser Tyr Ala Asp Asp Gly
Ala Leu Phe Asn Ile 1 5 10 465PRTArtificial SequenceSynthetic 46Ser
His Trp Met Ser 1 5 4716PRTArtificial SequenceSynthetic 47Ile Ile
Ala Ala Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15
4812PRTArtificial SequenceSynthetic 48Asp Tyr Gly Asp Tyr Arg Leu
Val Thr Phe Asn Ile 1 5 10 4937DNAArtificial SequenceSynthetic
49gaggaggagc tcactcccag tcggtgaagg agtccga 375059DNAArtificial
SequenceSynthetic 50ccgatgggcc cttggtggag gctgaggaga tggtgaccag
ggtgcctggt ccccagatg 595160DNAArtificial SequenceSynthetic
51gaggagaagc ttgttgctct ggatctctgg tgcctacggg gaactcgtgc tgactcagtc
605236DNAArtificial SequenceSynthetic 52attggatgca taatagatca
gtagcttggg aggctg 365330DNAArtificial SequenceSynthetic
53aaccagggca gcctcccaag ctactgatct 30
* * * * *