U.S. patent application number 15/970352 was filed with the patent office on 2018-09-06 for anti-human folate receptor beta antibodies and methods of use.
This patent application is currently assigned to PURDUE RESEARCH FOUNDATION. The applicant listed for this patent is PURDUE RESEARCH FOUNDATION, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY DEPARTMENT OF HE, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY DEPARTMENT OF HE. Invention is credited to Dimiter S. DIMITROV, Yang FENG, Philip S. LOW, Jiayin SHEN.
Application Number | 20180251544 15/970352 |
Document ID | / |
Family ID | 45811170 |
Filed Date | 2018-09-06 |
United States Patent
Application |
20180251544 |
Kind Code |
A1 |
LOW; Philip S. ; et
al. |
September 6, 2018 |
ANTI-HUMAN FOLATE RECEPTOR BETA ANTIBODIES AND METHODS OF USE
Abstract
Human anti-human folate receptor beta antibodies and
antigen-binding fragments thereof are described, as well as method
of using such antibodies and fragments to treat inflammatory
disorders or cancers expressing cell surface FR.beta..
Inventors: |
LOW; Philip S.; (West
Lafayette, IN) ; DIMITROV; Dimiter S.; (Frederick,
MD) ; FENG; Yang; (Frederick, MD) ; SHEN;
Jiayin; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PURDUE RESEARCH FOUNDATION
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY
DEPARTMENT OF HE |
West Lafayette
Rockville |
IN
MD |
US
US |
|
|
Assignee: |
PURDUE RESEARCH FOUNDATION
West Lafayette
IN
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY
DEPARTMENT OF HE
Rockville
MD
|
Family ID: |
45811170 |
Appl. No.: |
15/970352 |
Filed: |
May 3, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15094211 |
Apr 8, 2016 |
9963505 |
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15970352 |
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14524304 |
Oct 27, 2014 |
9914773 |
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15094211 |
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13790881 |
Mar 8, 2013 |
8871206 |
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14524304 |
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PCT/US2011/050943 |
Sep 9, 2011 |
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13790881 |
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61381340 |
Sep 9, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/732 20130101;
C07K 2317/54 20130101; A61K 47/6849 20170801; A61K 9/127 20130101;
C07K 2317/565 20130101; C07K 2317/76 20130101; A61K 47/6913
20170801; A61K 49/16 20130101; A61K 51/1027 20130101; C07K 14/705
20130101; C07K 2317/33 20130101; C07K 2317/92 20130101; A61K 9/0019
20130101; C07K 2317/21 20130101; A61K 49/0058 20130101; A61P 29/00
20180101; A61P 35/00 20180101; A61K 38/00 20130101; C07K 16/28
20130101; A61K 47/6911 20170801 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 9/00 20060101 A61K009/00; A61K 47/69 20060101
A61K047/69; A61K 47/68 20060101 A61K047/68; A61K 9/127 20060101
A61K009/127; A61K 38/00 20060101 A61K038/00; A61K 49/00 20060101
A61K049/00; A61K 49/16 20060101 A61K049/16; A61K 51/10 20060101
A61K051/10; C07K 14/705 20060101 C07K014/705 |
Claims
1. An isolated human monoclonal antibody, or an antigen-binding
fragment thereof, that specifically binds human folate receptor
beta (FR.beta.).
2. The antibody or fragment of claim 1 comprising: a heavy chain
variable region (V.sub.H) complementarity determining region (CDR)
1 comprising the amino acid sequence set forth in SEQ ID NO:1; a
V.sub.H CDR2 comprising the amino acid sequence set forth in SEQ ID
NO:2; and a V.sub.H CDR3 comprising the amino acid sequence set
forth in SEQ ID NO:3, wherein the antibody or fragment has one or
more properties selected from the group consisting of: (a) the
antibody or fragment does not detectably bind to human folate
receptor alpha (FR.alpha.); (b) the antibody or fragment binds to
human macrophages but not to mouse macrophages; (c) the antibody or
fragment has a binding affinity (EC.sub.50) of 20 nM; (d) the
antibody or fragment has a dissociation constant (Kd) of 6.39 nM;
and (e) the antibody mediates antibody-dependent cellular
cytotoxicity (ADCC) of FR.beta.-expressing target cells.
3. The antibody or fragment of claim 2, wherein said antibody or
fragment binds to cell surface FR.beta..
4. The antibody or fragment of claim 2, wherein said antibody is an
IgG1 antibody.
5. The antibody or fragment of claim 2, wherein said antibody or
fragment is de-fucosylated.
6. The antibody or fragment of claim 2, wherein the fragment is a
Fab antibody fragment, a F(ab').sub.2 fragment, or a single chain
antibody fragment (scFv).
7. The antibody or fragment of claim 2 conjugated with a
pharmaceutical agent.
8. The antibody or fragment of claim 7, wherein said pharmaceutical
agent is a chemotherapeutic.
9. The antibody or fragment of claim 2 conjugated to a
liposome.
10. The antibody or fragment of claim 9, wherein said liposome
comprises a pharmaceutical agent.
11. The antibody or fragment of claim 2 linked to a toxin.
12. The antibody or fragment of claim 11 wherein said antibody or
fragment thereof is covalently linked to said toxin.
13. The antibody or fragment of claim 2 linked to a detectable
moiety.
14. The antibody or fragment of claim 13, wherein said detectable
moiety is selected from the group consisting of a fluorescent
moiety, a luminescent moiety, a radioactive moiety, a CT contrast
agent, an MRI contrast agent, and biotin.
15. The antibody or fragment of claim 2, wherein the antibody or
fragment comprises one or more framework regions in SEQ ID
NO:7.
16. The antibody or fragment of claim 2, wherein the antibody or
fragment comprises SEQ ID NO:10.
17. The isolated antibody or fragment of claim 2, wherein the
antibody or fragment comprises: a light chain variable region
(V.sub.L) CDR1 comprising the amino acid sequence set forth in SEQ
ID NO:4; a V.sub.L CDR2 comprising the amino acid sequence set
forth in SEQ ID NO:5; and a V.sub.L CDR3 comprising the amino acid
sequence set forth in SEQ ID NO:6.
18. The isolated antibody or fragment of claim 2, wherein the
antibody or fragment comprises: a light chain variable region
(V.sub.L) CDR1 comprising the amino acid sequence set forth in SEQ
ID NO:14; a V.sub.L CDR2 comprising the amino acid sequence set
forth in SEQ ID NO:15; and a V.sub.L CDR3 comprising the amino acid
sequence set forth in SEQ ID NO:16.
19. An isolated human monoclonal antibody, or an antigen-binding
fragment thereof, that specifically binds human FR.beta., the
antibody or fragment comprising: a heavy chain variable region
(V.sub.H) CDR 1 comprising the amino acid sequence set forth in SEQ
ID NO:1; a V.sub.H CDR2 comprising the amino acid sequence set
forth in SEQ ID NO:2; a V.sub.H CDR3 comprising the amino acid
sequence set forth in SEQ ID NO:3; a light chain variable region
(V.sub.L) CDR1 comprising the amino acid sequence set forth in SEQ
ID NO:14; a V.sub.L CDR2 comprising the amino acid sequence set
forth in SEQ ID NO:15; and a V.sub.L CDR3 comprising the amino acid
sequence set forth in SEQ ID NO:16.
20. An isolated human monoclonal antibody, or an antigen-binding
fragment thereof, that specifically binds human FR.beta., the
antibody or fragment comprising: a heavy chain variable region
(V.sub.H) CDR1 comprising the amino acid sequence set forth in SEQ
ID NO:1; a V.sub.H CDR2 comprising the amino acid sequence set
forth in SEQ ID NO:2; a V.sub.H CDR3 comprising the amino acid
sequence set forth in SEQ ID NO:3; a light chain variable region
(V.sub.L) CDR1 comprising the amino acid sequence set forth in SEQ
ID NO:4; a V.sub.L CDR2 comprising the amino acid sequence set
forth in SEQ ID NO:5; and a V.sub.L CDR3 comprising the amino acid
sequence set forth in SEQ ID NO:6.
21. A composition comprising the antibody or fragment of claim 1
and a pharmaceutically acceptable carrier.
22. A method of treating a patient having an inflammatory disorder,
said method comprising administering to said patient, an amount of
the composition of claim 21 effective to reduce the number of
FR.beta. positive macrophages and monocytes in said patient.
Description
SEQUENCE LISTING
[0001] A sequence listing in electronic (ASCII text file) format is
filed with this application and incorporated herein by reference.
The name of the ASCII text file is "AttachE_Sequence_Listing.txt";
the file was created on Apr. 8, 2016; the size of the file is 17
KB.
TECHNICAL FIELD
[0002] This invention relates to anti-folate receptor beta
(FR.beta.) antibodies, and more particularly to human monoclonal
antibodies, or antigen-binding fragments thereof, that specifically
bind human FR.beta. and methods of using such antibodies or antigen
binding fragments thereof to reduce the number of FR.beta. positive
cells in subjects.
BACKGROUND
[0003] Folic acid is a vitamin required for the synthesis of
nucleotide bases and is essential for the proliferation of all
cells. Folates also are required for production of
S-adenosylmethionine, the common substrate used in methylation of
DNA, histones, G proteins, and many metabolic building blocks (see
Kim, J Nutr 135:2703-2709 (2005); Loenen, Biochem Soc Trans
34:330-333 (2006)). Almost all cells take in folic acid via the
reduced folate carrier or proton coupled folate transporter (see
Matherly and Goldman, Vitam Horm 66:403-456 (2003)). Some cells,
however, also express a folate receptor (FR) that binds folic acid
.about.100,000 times tighter than the aforementioned transporters,
and carries bound folates into cells by receptor-mediated
endocytosis (see Nakashima-Matsushita et al., Arth Rheum
42:1609-1616 (1999); Turk et al., Arthritis Rheumatoid 46:1947-1955
(2002)). There are four members of the FR family: FR.alpha.,
FR.beta., FR.gamma. and FR.delta. (see Elnakat and Ratnam, Adv Drug
Deliv Rev 56:1067-1084 (2004)). Different isoforms of the FR are
used by certain cancer cells, activated macrophages, and the
proximal tubule cells of the kidney to capture folates from their
environment (see e.g., Nakashima-Matsushita et al. 1999, supra; and
Turk et al. 2002, supra). A need exists for reagents and methods
for differential targeting of the folate receptors for treatment of
disease.
SUMMARY
[0004] This document is based on, inter alia, the identification of
a human monoclonal antibody that specifically binds FR.beta.. Such
an antibody or an antigen binding fragment thereof can be used to
reduce the number of FR.beta. positive cells in a subject, and is
useful for treating inflammatory disorders and cancers that express
cell surface FR.beta.. The antibody described herein is
particularly useful for treating human subjects as it is fully
human, with reduced risk for inducing immune responses and side
effects compared with murine, chimeric, or humanized
antibodies.
[0005] In one aspect, this document features an isolated human
monoclonal antibody, or an antigen-binding fragment thereof (e.g.,
Fab, a F(ab').sub.2 fragment, or a single chain antibody fragment
(scFv)) that specifically binds human folate receptor beta
(FR.beta.). The antibody or fragment can include a heavy chain
variable region (V.sub.H) complementarity determining region (CDR)
1 comprising the amino acid sequence set forth in SEQ ID NO:1; a
V.sub.H CDR2 comprising the amino acid sequence set forth in SEQ ID
NO:2; a V.sub.H CDR3 comprising the amino acid sequence set forth
in SEQ ID NO:3; a light chain variable region (V.sub.L) CDR1
comprising the amino acid sequence set forth in SEQ ID NO:4; a
V.sub.L CDR2 comprising the amino acid sequence set forth in SEQ ID
NO:5; and a V.sub.L CDR3 comprising the amino acid sequence set
forth in SEQ ID NO:6. The antibody or fragment can include one or
more framework regions in SEQ ID NO: 7 and SEQ ID NO:8. The
antibody or fragment can have the amino acid sequence set forth in
SEQ ID NO:10 or SEQ ID NO:11. The antibody or fragment can have the
amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8. The
antibody or fragment can bind to cell surface FR.beta.. The
antibody can be an IgG.sub.1 antibody. The antibody or fragment can
induce antibody-dependent cell-mediated cytotoxicity (ADCC) of
FR.beta. expressing target cells. In some embodiments, the antibody
or fragment is de-fucosylated. In some embodiments, the antibody or
fragment does not detectably bind to human folate receptor gamma
(FR.gamma.) or human folate receptor delta (FR.delta.).
[0006] In another aspect, this document features an isolated human
monoclonal antibody, or an antigen-binding fragment thereof (e.g.,
Fab, a F(ab').sub.2 fragment, or a scFv) that specifically binds
human FR.beta.. The antibody or fragment can include a V.sub.H CDR
1 comprising the amino acid sequence set forth in SEQ ID NO:1; a
V.sub.H CDR2 comprising the amino acid sequence set forth in SEQ ID
NO:2; a V.sub.H CDR3 comprising the amino acid sequence set forth
in SEQ ID NO:3; a V.sub.L CDR1 comprising the amino acid sequence
set forth in SEQ ID NO:14; a V.sub.L CDR2 comprising the amino acid
sequence set forth in SEQ ID NO:15; and a V.sub.L CDR3 comprising
the amino acid sequence set forth in SEQ ID NO:16. The antibody or
fragment can include one or more framework regions in SEQ ID NO: 7
and SEQ ID NO: 17. The antibody or fragment can have the amino acid
sequence set forth in SEQ ID NO:10 or SEQ ID NO:18. The antibody or
fragment can have the amino acid sequence set forth in SEQ ID NO:7
or SEQ ID NO:17. The antibody or fragment can bind to cell surface
FR.beta.. The antibody can be an IgG.sub.1 antibody. The antibody
or fragment can induce ADCC of FR.beta. expressing target cells. In
some embodiments, the antibody or fragment is de-fucosylated. In
some embodiments, the antibody or fragment does not detectably bind
to human FR.gamma. or human FR.delta..
[0007] This document also features an isolated human monoclonal
antibody, or an antigen-binding fragment thereof (e.g., Fab, a
F(ab').sub.2 fragment, or a scFv) that specifically binds human
FR.beta.. The antibody or fragment contains a V.sub.H CDR 1
comprising the amino acid sequence set forth in SEQ ID NO:1; a
V.sub.H CDR2 comprising the amino acid sequence set forth in SEQ ID
NO:2; and a V.sub.H CDR3 comprising the amino acid sequence set
forth in SEQ ID NO:3. The antibody or fragment has one or more
properties selected from the group consisting of: (a) the antibody
or fragment does not detectably bind to human folate receptor alpha
(FR.alpha.); (b) the antibody or fragment binds to human
macrophages but not to mouse macrophages; (c) the antibody or
fragment has a binding affinity (EC.sub.50) of 20 nM; (d) the
antibody or fragment has a dissociation constant (Kd) of 6.39 nM;
and (e) the antibody mediates ADCC of FR.beta.-expressing target
cells. In some embodiments, the antibody or fragment does not
detectably bind to human FR.gamma. or human FR.delta..
[0008] This document also features an isolated heavy chain of an
antibody, or antigen binding fragment of an antibody, that
specifically binds human FR.beta.. The isolated heavy chain
comprises a V.sub.H CDR 1 comprising the amino acid sequence set
forth in SEQ ID NO:1; a V.sub.H CDR2 comprising the amino acid
sequence set forth in SEQ ID NO:2; and a V.sub.H CDR3 comprising
the amino acid sequence set forth in SEQ ID NO:3, wherein the
antibody or fragment has one or more properties selected from the
group consisting of: (a) the antibody or fragment does not
detectably bind to human FR.alpha.; (b) the antibody or fragment
binds to human macrophages but not to mouse macrophages; (c) the
antibody or fragment has a binding affinity (EC.sub.50) of 20 nM;
(d) the antibody or fragment has a dissociation constant (Kd) of
6.393 nM; and (e) the ADCC of FR.beta.-expressing target cells. In
some embodiments, the antibody or fragment does not detectably bind
to human FR.gamma. or human FR.delta..
[0009] An antibody or fragment described herein can be conjugated
with a pharmaceutical agent (e.g., a chemotherapeutic) or a
liposome (e.g., a liposome including a pharmaceutical agent),
linked to a toxin (e.g., covalently linked to the toxin) or a
detectable moiety. The detectable moiety can be selected from the
group consisting of a fluorescent moiety, a luminescent moiety, a
radioactive moiety, a CT contrast agent, an MRI contrast agent, and
biotin.
[0010] This document also features compositions including any of
the antibodies or fragments described herein and a pharmaceutically
acceptable carrier, and methods of using such compositions. For
example, a composition can be used in a method of treating a
patient having an inflammatory disorder (e.g., atherosclerosis,
ischemia/reperfusion injury, transplantation rejection, vasculitis,
inflammatory osteoarthritis, glomerulonephritis, restenosis,
systemic sclerosis, fibromyalgia, sarcoidosis, or an autoimmune
disease such as rheumatoid arthritis, systemic lupus erythematosus
(SLE), ulcerative colitis, psoriasis, Type 1 diabetes, Crohn's
disease, multiple sclerosis, and Sjogren's disease). The method can
include administering to the patient an amount of a composition
effective to reduce the number of FR.beta. positive macrophages and
monocytes in the patient.
[0011] A composition described herein also can be used in a method
of treating a patient having a cancer expressing cell-surface
FR.beta.. The method can include administering to the patient, an
amount of the composition effective to reduce the number of
FR.beta. expressing cancer cells in the patient. The cancer can be
a myeloid cancer (e.g., acute myeloid leukemia or chronic myeloid
leukemia), multiple myeloma, or a solid FR.beta. expressing cancer
(e.g., a non-epithelial malignancy).
[0012] This document also features a method of depleting activated
macrophages from a human subject. The method includes administering
to the subject a human monoclonal antibody, or antigen-binding
fragment thereof, that specifically binds human FR.beta., in an
amount effective to reduce the number of activated macrophages in
the subject.
[0013] In any of the methods described herein, the antibody or
fragment thereof can induce ADCC of FR.beta. expressing target
cells. In some embodiments, the antibody or fragment is
de-fucosylated.
[0014] In any of the methods described herein, the antibody or
fragment can induce opsonization-mediated clearance of FR.beta.
expressing target cells.
[0015] In any of the methods described herein, the antibody or
fragment can induce complement-mediated lysis of FR.beta.
expressing target cells.
[0016] This document also features a nucleic acid that includes
first and second segments, the first segment encoding a scFv
fragment of a human monoclonal antibody that specifically binds
human FR.beta., and the second segment encoding at least the
intracellular domain of a T cell signaling polypeptide. The T cell
signaling polypeptide is a CD3 zeta chain or FcR.gamma. chain. The
nucleic acid further can include a third segment encoding at least
the intracellular domain of a costimulatory polypeptide (e.g., a
costimulatory polypeptide selected from the group consisting of
CD28, CD40L, CD134, CD137, and PD-1). In addition, this document
features cells that include any such nucleic acids. The cells can
be T cells (e.g., cytotoxic T cells, CD8+ T cells, or CD4+ T
cells). The T cells can produce at least one cytokine or lymphokine
(e.g., interferon-.gamma. (IFN-.gamma.)).
[0017] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0018] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0019] FIG. 1 is a depiction of the amino acid sequence of the
full-length, unprocessed human FR.beta. protein (SEQ ID NO:9). A
fragment of the full-length protein containing residues 22 to 236
was used to produce recombinant FR.beta..
[0020] FIG. 2A is a depiction of the amino acid sequence of the
m909 Fab heavy chain sequence (SEQ ID NO:7) and FIG. 2B is a
depiction of the amino acid sequence of the m909 Fab light chain
lambda sequence (SEQ ID NO:17). In each of the sequences, the
framework regions are italicized, the CDRs are underlined, and the
constant region is in lowercase, non-italicized text. Framework
regions 1-4 (FR1-FR4) are numbered consecutively from the
N-terminus, with FR1 being the most N-terminal sequence. CDRs 1-3
also are numbered consecutively from the N-terminus, with CDR1
being the most N-terminal.
[0021] FIG. 3 is a graph showing the binding of m909 IgG to
FR.beta..
[0022] FIG. 4 is a one-dimensional fluorescent flow cytometry (FFC)
histogram showing that bivalency allows binding of scFv m909 to
FR.beta. positive cells.
[0023] FIG. 5 is a one-dimensional FFC histogram showing that scFv
m909 does not bind to FR.beta. negative cells.
[0024] FIGS. 6A and 6B depict isoform-specific binding of
FITC-human anti-human FR.beta. mAb to FR.beta.-positive/negative
cell lines. FIG. 6A contains four one-dimensional FFC histograms of
control FITC-isotype IgG1 binding to CHO-hFR.beta. (a) or
FITC-human anti-human FR.beta. IgG1 binding to CHO-hFR.beta. cells
(b), KB cells (c), or CHO-K1 cells. FIG. 6B is a line graph showing
the concentration of fluorescent intensity vs. human anti-human
FR.beta.IgG1.
[0025] FIG. 7 is a bar graph showing the percent lysis of preB L1.2
FR.beta.-positive cells at various concentrations of IgG.sub.1 m909
or control IgG.sub.1.
[0026] FIG. 8 contains a bar graph of the percent lysis of
CHO-FR.beta. cells (left panel) and a bar graph of the percent
lysis of CHO-K1 cells (right panel).
[0027] FIG. 9A contains representative 2-dimensional and
1-dimensional FFC analyses of FITC-anti-FR.beta. m909 IgG1 binding
to bacteria-recruited peritoneal FR.beta.+ macrophages. In a),
FR.beta.+ macrophages were stained with folate conjugated to FITC.
In b), FR.beta.+ macrophages were stained with FITC conjugated to
human anti-human FR.beta. m909 IgG1. The percentage of FR.beta.+
cells within each gate shown in the left hand two panels
(2-dimensional FFC histograms) are shown in the right hand two
panels (1-dimensional FFC histograms).
[0028] FIG. 9B contains representative 2-dimensional and
one-dimensional FFC analyses of FITC-anti-FR.beta. m909 IgG1
binding to the FR.beta.+CD14.sup.High CD16.sup.- subset of human
peripheral blood monocytes. The populations of cells gated in the
left hand 2-dimensional histograms were subjected to analysis using
fluorochrome-conjugated anti-CD16 and anti-CD14 antibodies (middle
two 2-dimensional FFC histograms) and the percentage of FR.beta.+
cells in the gated populations in the latter histograms were
determined (right hand 1-dimensional FFC histograms).
DETAILED DESCRIPTION
[0029] In general, this document provides human anti-human FR.beta.
monoclonal antibodies or antigen-binding fragments thereof, as well
as methods of using such antibodies or fragments thereof to treat,
detect, or monitor an inflammatory disorder or a cancer expressing
FR.beta. in a patient (e.g., a human patient). The term "FR.beta."
as used herein refers to human folate receptor beta. The amino acid
sequence of human FR.beta. can be found in FIG. 1 (SEQ ID NO:9) and
in GenBank under Accession No. NP 001107007. FR.beta. is a
differential marker on the surface of myelomonocytic lineage cells.
In normal tissues, FR.beta. is expressed in placenta,
myelomonocytic lineage cells (e.g., monocytes and macrophages), and
mature neutrophils. FR.beta., however, does not bind folic acid on
quiescent macrophages until the myeloid cell becomes activated.
FR.beta. is consistently detected in multiple myeloma cells,
chronic myeloid leukemia (CML) cells, and in 70% of acute myeloid
leukemia (AML) cells. See, for example, Nakashima-Matsushita et
al., 1999, supra; and Ross et al., Contrast Media Mol Imaging
2:72-81 (1999). FR.beta. also has been detected by RT-PCR in solid
tumors (e.g., carcinomas from colon, kidney, breast, ovary, uterus,
or lung; squamous cell carcinomas of the head and neck; and
malignancies of non-epithelial origin such as sarcomas, lymphomas,
fibrous histiocytomas, ovarian granulosa cell tumor, astrocytoma,
meningiomas, and Wilms' tumor). See, for example, Ross et al.,
Cancer, 73:2432-43 (1994).
[0030] In monocytes and macrophage-lineage cells, FR.beta.
expression is increased upon activation. For example, FR.beta. is
expressed and functional in synovial macrophages in rheumatoid
arthritis patients. Furthermore, .gamma.-scintigraphy images of
patients with a variety of inflammatory disorders (e.g., rheumatoid
arthritis, Crohn's disease, ischemic bowel disease, Sjogren's
syndrome, localized infections, atherosclerosis, and organ
transplant rejection) show uptake of a folate-targeted .sup.99mTc
imaging agent (EC20) at sites of inflammation (see Low et al., Acc
Chem Res. 41(1):120-9 (2008); Matteson et al., Clin Exp Rheumatol
27:253-259 (2009); and Ayala-Lopez et al., J Nuc Med, 51:768-774
(2010)). These findings indicate expression of FR.beta. in
activated macrophages and monocytes.
[0031] The antibodies and antigen binding fragments described
herein specifically bind FR.beta., and do not detectably bind FRI
In some embodiments, the antibodies and antigen binding fragments
described herein also do not detectably bind FRK and/or FR.LAMBDA..
FRa is expressed on the apical surfaces of a few epithelial cell
types (primarily proximal tubules of the kidneys and alveolar
epithelial cells of the lungs) and is upregulated on a variety of
epithelial-derived tumors (see Weitman et al., Cancer Res
52:6708-6711 (1992); Salazar and Ratnam, Cancer Mestastasis Rev
26:141-152 (2007); and Leamon and Jackman, Vitam Horm 79:203-233
(2008)). FR.gamma. is rarely expressed and difficult to detect in
vivo. FR.delta. is expressed on regulatory T cells, where it
exhibits only very low affinity for folic acid. As such, the
antibodies and antigen binding fragments described herein, which
specifically bind FR.beta., can be used for depleting activated
macrophages, treating inflammatory disorders, and treating cancers
that express FR.beta..
Human Anti-Human FR.beta. Antibodies
[0032] This document features fully human monoclonal antibodies
(mAb), or antigen-binding fragments thereof, that specifically bind
human FR.beta.. As used herein, the term "antibody" refers to a
protein that generally comprises heavy chain polypeptides and light
chain polypeptides. IgG, IgD, and IgE antibodies comprise two heavy
chain polypeptides and two light chain polypeptides. IgA antibodies
comprise two or four of each chain and IgM generally comprise 10 of
each chain. Single domain antibodies having one heavy chain and one
light chain and heavy chain antibodies devoid of light chains are
also contemplated. A given antibody comprises one of five types of
heavy chains, called alpha (1 or 2), delta, epsilon, gamma (1, 2,
3, or 4), and mu, the categorization of which is based on the amino
acid sequence of the heavy chain constant region. These different
types of heavy chains give rise to five classes of antibodies, IgA
(including IgA1 and IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3 and
IgG4) and IgM, respectively.
[0033] In an IgG antibody, the heavy chain includes a "hinge
region", "C.sub.H1 region", "C.sub.H2 region", "C.sub.H3 region"
each of which refers to a different region of the heavy chain
constant region. The hinge region is located between the C.sub.H1
region and C.sub.H2 region, and links the Fab region to the Fc
region. Thus, N terminal to C terminal, the regions are in the
order C.sub.H1, hinge, C.sub.H2, and C.sub.H3. In the .mu. heavy
chain constant regions of IgM antibodies, there is an extra
C-terminal region, the C.sub.H4 region. The antibodies and
fragments of this document can contain all or some of the regions
of the heavy chain constant regions. Thus, they can contain all or
part or the hinge region, and/or all or part of the C.sub.H1
region, and/or all or part of the C.sub.H2 region, and/or all or
part of the C.sub.H3 region, and/or all or part of the C.sub.H4
region. Moreover, any of these regions, or parts of regions, can in
any particular antibody or fragment, be derived from one, two,
three, four, five, six, seven, eight, or nine different constant
region classes or subclasses (K1, 2, 3, or 4, I1 or 2, M, .mu., and
A).
[0034] Antibodies and antigen binding fragments described herein
can be of any class and have the corresponding heavy chain constant
region. A given antibody also comprises one of two types of light
chains, called kappa or lambda, the categorization of which is
based on the amino acid sequence of the light chain constant
regions. Antibodies and antigen binding fragments described herein
can have either class of light chain.
[0035] Fully human monoclonal antibodies that specifically bind to
FR.beta. can be produced, e.g., using in vitro-primed human
splenocytes as described by Boerner et al., J. Immunol., 147, 86-95
(1991), or repertoire cloning as described by Persson et al., Proc.
Nat. Acad. Sci. USA, 88: 2432-2436 (1991); Huang and Stollar, J.
Immunol. Methods 141, 227-236 (1991); or U.S. Pat. No. 5,798,230.
Large nonimmunized or immunized human phage display libraries also
can be used to isolate high affinity antibodies using standard
phage technology (see, e.g., Hoogenboom et al., Immunotechnology
4:1-20 (1998); Hoogenboom et al., Immunol Today 2:371-8 (2000); and
U.S. Patent Publication No. 2003-0232333). In addition, a
TransChromo mouse carrying human immunoglobulin loci, including all
the subclasses of IgGs (IgG1-G4), can be used to prepare human
monoclonal antibodies. See, Ischida et al., Cloning Stem Cells
4(1):91-102 (2002).
[0036] "Antigen binding fragment" of an antibody as the term is
used herein refers to an antigen binding molecule that is not a
complete antibody as defined above, but that still retains at least
one antigen binding site. Thus, in one embodiment, an antigen
binding fragment is a fragment of an antibody that binds to
FR.beta.. Antibody fragments often include a cleaved portion of a
whole antibody, although the term is not limited to such cleaved
fragments. Antigen binding fragments can include, for example, a
Fab, F(ab').sub.2, Fv, and single chain Fv (scFv) fragment. An scFv
fragment is a single polypeptide chain that includes both the heavy
and light chain variable regions of the antibody from which the
scFv is derived. Other suitable antibodies or antigen binding
fragments include linear antibodies, multispecific antibody
fragments such as bispecific, trispecific, and multispecific
antibodies (e.g., diabodies (Poljak Structure 2(12):1121-1123
(1994); Hudson et al., J. Immunol. Methods 23(1-2):177-189 (1994)),
triabodies, tetrabodies), minibodies, chelating recombinant
antibodies, intrabodies (Huston et al., Hum. Antibodies
10(3-4):127-142 (2001); Wheeler et al., Mol. Ther. 8(3):355-366
(2003); Stocks Drug Discov. Today 9(22): 960-966 (2004)),
nanobodies, small modular immunopharmaceuticals (SMIP),
binding-domain immunoglobulin fusion proteins, camelid antibodies,
camelized antibodies, and V.sub.HH containing antibodies.
[0037] A human anti-human FR.beta. mAb or antigen-binding fragment
thereof described herein specifically binds to human FR.beta., but
does not detectably bind to FRI. In some embodiments, the
anti-human FR.beta. mAb or antigen-binding fragment thereof also
does not detectably bind to FRK and/or FRA. Binding specificity can
be assessed as described herein. For example, binding specificity
of an antibody or antigen binding fragment thereof can be assessed
using recombinantly produced FR or cells expressing a particular FR
on their cell surface and techniques such as enzyme linked
immunosorbent assay (ELISA) or flow cytometry. As described herein,
human anti-human FR.beta. mAb can bind cell surface FR.beta., and
antigen binding fragments of human anti-human FR.beta. mAb (e.g.,
Fab or scFv) can bind cell surface FR.beta. mAb when bivalent.
Human anti-human FR.beta. mAb can bind to human macrophages but not
to mouse macrophages. As described herein, in Fab format, m909
bound to recombinant FR.beta. with high (nM) affinity (EC.sub.50=20
nM). In an IgG1 format, m909 has high (subnanomolar) avidity in
vitro and specifically binds to cell surface FR.beta.. As described
in Example 3, the human anti-human FR.beta. mAb has a dissociation
constant (Kd) of 6.393 nM.
[0038] In some embodiments, an antibody or antigen binding fragment
thereof described herein contains a heavy chain variable region
(V.sub.H) complementarity determining region (CDR) 1 having the
amino acid sequence set forth in SEQ ID NO:1 [GYTFTSYA], a V.sub.H
CDR2 having the amino acid sequence set forth in SEQ ID NO:2
[KYSQKFQ]; and a V.sub.H CDR3 having the amino acid sequence set
forth in SEQ ID NO:3 [ARDISYGSFDYW]. Alternatively, or in addition,
the antibody or antigen binding fragment thereof described herein
contains a light chain variable region (V.sub.L) CDR1 having the
amino acid sequence set forth in SEQ ID NO:4 [NLRSYY]; a V.sub.L
CDR2 having the amino acid sequence set forth in SEQ ID NO:5 [GKN];
and a V.sub.L CDR3 having the amino acid sequence set forth in SEQ
ID NO:6 [HSRKSRGNHLLF].
[0039] In some embodiments, the antibody or fragment described
herein can include one or more framework regions italicized in the
following amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 7
[EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMG
WINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARD
ISYGSFDYWGQGTLVTVSSastkgpsvfplapsskstsggtaalgclvkd
yfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqty
icnvnhkpsntkvdkkvepkscdktsgqag],
heavy chain sequence; constant region in lower class letters)).
Alternatively, or in addition, the antibody or fragment described
herein can include one or more framework regions italicized in the
following amino acid sequence:
TABLE-US-00002 (SEQ ID NO: 8
[SSELTQDPAVSVALGQTVRITCQGDNLRSYYASWYRQKSGQAPVLVIYG
KNNRPSGIPDRFSGSSSGNTASLTITAAQAEDEADYYCHSRKSRGNHLLF
GGGTKLTVLgqpkaapsvtlfppsseelqankatlvclisdfypgavtva
kadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthe
gstvektvaptecs],
lambda light chain sequence; constant region in lower case
letters).
[0040] In some embodiments, the antibody or antigen binding
fragment thereof comprises the heavy chain variable region
(V.sub.H) amino acid sequence set forth in SEQ ID NO:10
[EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWI
NAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSF
DYWGQGTLVTVSS]. Alternatively, or in addition, the antibody or
antigen binding fragment thereof comprises the light chain variable
region amino (V.sub.1) amino acid sequence set forth in SEQ ID
NO:11
TABLE-US-00003 [SSELTQDPAVSVALGQTVRITCQGDNLRSYYASWYRQKSGQAPVLVIYG
KNNRPSGIPDRFSGSSSGNTASLTITAAQAEDEADYYCHSRKSRGNHLLF GGGTKLTVL].
In some embodiments, an antibody or fragment thereof comprising
such sequences is designated m923 and specifically binds to human
FR.beta..
[0041] In some embodiments, the antibody or fragment described
herein can include the following heavy chain amino acid sequence:
[EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWI
NAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSF
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTSGQAG] (SEQ ID NO:7). Alternatively, or in addition, the
antibody or fragment described herein can include the following
light chain amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 8)
[SSELTQDPAVSVALGQTVRITCQGDNLRSYYASWYRQKSGQAPVLVIYG
KNNRPSGIPDRFSGSSSGNTASLTITAAQAEDEADYYCHSRKSRGNHLLF
GGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA
KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE
GSTVEKTVAPTECS].
[0042] In some embodiments, the antibody or antigen binding
fragment thereof described herein contains a heavy chain variable
region (V.sub.H) complementarity determining region (CDR) 1 having
the amino acid sequence set forth in SEQ ID NO:1 [GYTFTSYA], a
V.sub.H CDR2 having the amino acid sequence set forth in SEQ ID
NO:2 [KYSQKFQ]; and a V.sub.H CDR3 having the amino acid sequence
set forth in SEQ ID NO:3 [ARDISYGSFDYW]. Alternatively, or in
addition, the antibody or antigen binding fragment thereof
described herein contains a light chain variable region (V.sub.L)
CDR1 having the amino acid sequence set forth in SEQ ID NO:14
[SLRSNY]; a V.sub.L CDR2 having the amino acid sequence set forth
in SEQ ID NO:15 [GQN]; and a V.sub.L CDR3 having the amino acid
sequence set forth in SEQ ID NO:16 [DSRVSTGNHVVF].
[0043] In some embodiments, an antibody or fragment described
herein can include one or more framework regions italicized in the
following amino acid sequence:
TABLE-US-00005 (SEQ ID NO: 7
[EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMG
WINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARD
ISYGSFDYWGQGTLVTVSSastkgpsvfplapsskstsggtaalgclvkd
yfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqty
icnvnhkpsntkvdkkvepkscdktsgqag],
heavy chain sequence; constant region in lower case letters).
Alternatively, or in addition, the antibody or fragment described
herein can include one or more framework regions italicized in the
following amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 17
[SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYG
QNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGNHVVF
GGGTKLTVLgqpkaapsvtlfppsseelqankatlvclisdfypgavtva
kadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthe
gstvektvaptecs],
lambda light chain sequence; constant region in lower case
letters).
[0044] In some embodiments, the antibody or antigen binding
fragment thereof comprises the V.sub.H amino acid sequence set
forth in SEQ ID NO:10
[EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNG
NTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVT VSS].
Alternatively, or in addition, the antibody or antigen binding
fragment thereof comprises the V.sub.L amino acid sequence set
forth in SEQ ID NO:18
[SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQNNRPSGIPD
RFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGNHVVFGGGTKLTVL]. In some
embodiments, the antibody or antigen binding fragment thereof is
designated m909.
[0045] In some embodiments, an antibody or fragment described
herein can include the following heavy chain amino acid sequence:
[EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWI
NAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSF
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTSGQAG] (SEQ ID NO:7). Alternatively, or in addition, the
antibody or fragment described herein can include the following
light chain amino acid sequence:
TABLE-US-00007 (SEQ ID NO: 17)
[SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIY
GQNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGNHV
VFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV
TVAKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ
VTHEGSTVEKTVAPTECS].
[0046] It is understood that antibodies and antigen binding
fragments described above, whether specified in terms of heavy and
light chain components, or heavy or light chain components, can
have any of the functional properties and activities described
herein.
[0047] In some embodiments, the full length heavy or light chain,
variable region, CDR, framework region, or constant region of an
antibody or antigen binding fragment described herein can have at
least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%,
98%, 99%, or 100% sequence identity) to the corresponding amino
acid sequence set forth in any one of SEQ ID NOs:1-8, 10, 11, and
14-18. The percent identity between a particular amino acid
sequence and the amino acid sequence set forth in any one of SEQ ID
NOs:1-8, 10, 11, and 14-18 can be determined as follows. First, the
amino acid sequences are aligned using the BLAST 2 Sequences
(Bl2seq) program from the stand-alone version of BLASTZ containing
BLASTP version 2.0.14. This stand-alone version of BLASTZ can be
obtained from Fish & Richardson's web site (e.g.,
www.fr.com/blast/) or the U.S. government's National Center for
Biotechnology Information web site (www.ncbi.nlm.nih.gov).
Instructions explaining how to use the Bl2seq program can be found
in the readme file accompanying BLASTZ. Bl2seq performs a
comparison between two amino acid sequences using the BLASTP
algorithm. To compare two amino acid sequences, the options of
Bl2seq are set as follows: -i is set to a file containing the first
amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set
to a file containing the second amino acid sequence to be compared
(e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired
file name (e.g., C:\output.txt); and all other options are left at
their default setting. For example, the following command can be
used to generate an output file containing a comparison between two
amino acid sequences: C:\Bl2seq c:\seq1.txt -j c:\seq2.txt -p
blastp -o c:\output.txt. If the two compared sequences share
homology, then the designated output file will present those
regions of homology as aligned sequences. If the two compared
sequences do not share homology, then the designated output file
will not present aligned sequences. Similar procedures can be
followed for nucleic acid sequences except that blastn is used.
[0048] Once aligned, the number of matches is determined by
counting the number of positions where an identical amino acid
residue is presented in both sequences. The percent identity is
determined by dividing the number of matches by the length of the
amino acid sequence in SEQ ID NOs:1-8, 10, 11, or 14-18, followed
by multiplying the resulting value by 100.
[0049] It is noted that the percent identity value is rounded to
the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is
rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19
is rounded up to 78.2. It also is noted that the length value will
always be an integer.
[0050] It will be appreciated that a number of nucleic acids can
encode the amino acid sequences set forth in SEQ ID NOs:1-8, 10,
11, and 14-18. The degeneracy of the genetic code is well known to
the art; i.e., for many amino acids, there is more than one
nucleotide triplet that serves as the codon for the amino acid. The
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7
is set forth in SEQ ID NO: 19. The nucleotide sequence encoding the
amino acid sequence of SEQ ID NO:8 is set forth in SEQ ID NO: 20.
The nucleotide sequence encoding the amino acid sequence of SEQ ID
NO: 17 is set forth in SEQ ID NO:21. In some embodiments, a heavy
or light chain of an antibody is encoded by a nucleic acid having
at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%,
98%, 99%, or 100% sequence identity) to the nucleotide sequences
set forth in SEQ ID NOs. 19-21. Sequence identity is calculated as
described above for protein sequences except that blastn is
used.
[0051] In some embodiments, an antibody or antigen binding fragment
thereof described herein induces antibody-dependent cell-mediated
cytotoxicity (ADCC) of FR.beta. expressing target cells. Suitable
antigen binding fragments that induce ADCC have a functional Fc
region, i.e., an Fc region that can bind to an Fc receptor on an
ADCC effector cell. The antibodies and antigen binding fragments
thereof can bind to IgG Fc binding receptors (Fc.gamma.R) receptors
I, II and III (CD64, CD32, and CD16) in order to mediate the
functional activities described herein. In some embodiments,
binding to a FcKR (e.g., FcKR III) can be enhanced by removing
fucose residues from and/or by increasing galactosylation of the
glycan present on the Fc portion of an IgG.sub.1. See, for example,
Houdes et al., Molecular & Cellular Proteomics 9:1716-1728
(2010); Kubota et al., Cancer Sci., 100: 1566-1572 (2009);
Malphettes et al., Biotechnol. Bioeng., 106:774-783 (2010); and
Raju, Curr. Opin. Immunol., 20:471-478 (2008). De-fucosylated
antibodies can be produced using, for example, cells with reduced
expression of the GDP-4,6-dehydratase gene (e.g., from a mutation
such as that in Chinese hamster ovary (CHO) Lec13 cells or from a
small interfering RNA against the GDP-4,6-dehydratase gene), cells
in which the I-1,6-fucosyltransferase (FUT8) has been knocked out
or expression reduced (e.g., using a small interfering RNA (siRNA)
against the FUT8 gene), cells co-expressing
.beta.-1,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi
I mannosidase II (ManII), or cells expressing
GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). See, Ishiguro et
al., Cancer Sci., pages 1-7, July, 2010; and von Horsten,
Glycobiology, published online Jul. 23, 2010. FcKR binding also can
be enhanced by mutating relevant amino acids in the heavy chain
constant regions comprising the Fc region (e.g., the hinge region,
the C.sub.H2 region, or the C.sub.H3 region). See, for example,
Natsume et al., Drug Des. Develop. Ther., 3:7-16 (2009). In some
embodiments, an antibody or fragment thereof can induce
opsonization-mediated clearance and/or induce complement-mediated
lysis of FR.beta. expressing target cells. ADCC can be assessed in
vitro using a lactate dehydrogenase (LDH) release assay or
chromium-51 release assay.
[0052] This document also provides antibodies that bind to
particular epitopes of FR.beta. that contain, for example, at least
three amino acids of human FR.beta. (FIG. 1, SEQ ID NO: 9). For
example, an epitope can contain 3 to 30 such as 5 to 25, 7 to 23,
10 to 20, or 13 to 18 amino acids of human FR.beta.). For example,
an epitope can contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino
acids of human FR.beta.. The epitope can be in the N-terminal half
(i.e., residues 1 to 127 of SEQ ID NO: 9) or the C-terminal half of
human FR.beta. (i.e., residues 128 to 255 of SEQ ID NO:9).
[0053] An antibody or antigen binding fragment described herein can
be conjugated or linked, either covalently or noncovalently, to a
variety of molecules, including pharmaceutical agents, liposomes,
oligonucleotides (e.g., small interfering RNA (siRNA)), toxins,
detectable moieties, or biological molecules (e.g., a cytokine such
as an interleukin (IL) 2, IL4, IL12, 13 or 15; interferon (IFN) I,
IFN .beta., or IFN K) using methods known in the art. Conjugating
or linking such molecules to an antibody or antigen binding
fragment described herein allows the molecule to be targeted
specifically to FR.beta.. As such, molecules can be delivered to
the desired site while minimizing toxicity.
[0054] For example, an antibody or antigen binding fragment can be
conjugated with a pharmaceutical agent such as a chemotherapeutic
(e.g., cisplatin, carboplatin, procarbazine, mechlorethamine,
cyclophosphamide, camptothecin, adriamycin, ifosfamide, melphalan,
chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,
doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil,
podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil,
vincristin, vinblastin, methotrexate, or an analog of any of the
aforementioned). An antibody or antigen binding fragment also can
be conjugated with an anti-inflammatory agent such as a
glucocorticoid, nonsteroidal anti-inflammatory agent,
phosphoinositide-3-kinase inhibitor (e.g., wortmannin or
derivatives such as demethoxyviridin, PX-866, LY294002, and
LY294002 RGDS (Arg-Gly-Asp-Ser)-conjugated pro-drug SF1126, see,
e.g., Maira et al., Biochem. Soc. Trans., 37, 265-272 (2009));
NF-kappa-B inhibitor, I-kappa-B kinase inhibitor, mTOR (mammalian
target of rapamycin) inhibitor (e.g., rapamycin, CCI-779, RAD001,
or AP23573, see Maira et al., supra), mitogen activated protein
(MAP) kinase inhibitor (e.g., SB-203580 and VX-745, see Brown et
al., J. Inflammation, 5:22 (2008), or a Janus kinase (JAK)
inhibitor.
[0055] An antibody or antigen binding fragment also can be
conjugated with a liposome. See, for example, the nanoliposomes of
Low et al. (Accounts of Chemical Research, 41(1):120-129 (2008))
that are less than 100 nm in diameter and contain a portion of
PEGylated lipids (i.e., a lipid linked to polyethylene glycol
(PEG)). Liposomes can be loaded with a pharmaceutical agent using
methods known in the art.
[0056] An antibody or antigen binding fragment described herein can
be linked to a toxin such as Pseudomonas exotoxin A (PE),
diphtheria toxin (DT), gelonin, saporin, ricin A, abrin, mistletoe
lectin, modeccin, pokeweed antiviral protein (PAP), Bryodin 1,
bouganin, or biologically active fragments thereof, to generate an
immunotoxin. See, for example, Kreitman, BioDrugs, 23(1):1-13
(2009). PE and DT, and biologically active fragments thereof, are
particularly useful. A biologically active fragment of PE can
include, for example, amino acids 253-364 and 381-613 of PE as
described by Hassan et al., J Immunother., 23:473-9 (2000). A
biologically active fragment of DT can include DT388 or DAB389,
which contain the first 388 or 389 amino acids of DT. See, for
example, Chaudhary et al., Biochem Biophys Res Commun., 180:545-51
(1991). Such immunotoxins are useful for killing FR.beta.
expressing cells (e.g., any of the cancer cells described herein)
in vivo or in vitro.
[0057] An antibody or antigen binding fragment described herein can
be linked to a detectable moiety. Suitable detectable moieties
include, without limitation, radionuclides (e.g., radionuclides
used for in vivo diagnostics such as .sup.186Re, .sup.188Re,
.sup.64Cu, .sup.67Cu, .sup.212Bi, .sup.123I, .sup.131I, .sup.211At,
.sup.177Lu, .sup.47Sc, .sup.105Rh, .sup.109Pd, .sup.153Sm,
.sup.199Au, .sup.99mTc, .sup.111In, .sup.124I, .sup.18F, .sup.11C,
.sup.198Au, .sup.75Br, .sup.76Br, .sup.77Br, .sup.13N, .sup.34mCl,
.sup.38Cl, .sup.52mMn, .sup.55Co, .sup.62Cu, .sup.68Ga, .sup.72As,
.sup.76As, .sup.72Se, .sup.73Se, or .sup.75Se; or radionuclides
useful for in vitro experiments such as .sup.125I, .sup.35S,
.sup.3H, .sup.32P, .sup.33P, or .sup.14C), fluorescent moieties
(e.g., fluorescein, fluorescein isothiocyanate (FITC), PerCP,
rhodamine, or phycoerythrin (PE)), luminescent moieties (e.g.,
Qdot.TM. nanoparticles supplied by the Quantum Dot Corporation,
Palo Alto, Calif.), compounds that absorb light of a defined
wavelength, or enzymes (e.g., alkaline phosphatase or horseradish
peroxidase). Antibodies can be indirectly labeled by conjugation
with biotin then detected with avidin or streptavidin labeled with
a molecule described above. Methods of detecting or quantifying a
detectable moiety depend on the nature of the moiety and are known
in the art. Examples of detectors include, without limitation,
x-ray film, radioactivity counters, scintillation counters,
spectrophotometers, colorimeters, fluorometers, luminometers, and
densitometers. Methods of attaching the radionuclide atoms (or
larger molecules/chelates containing them) to an antibody or
antigen binding fragment thereof are known in the art and can
include incubating the antibody or fragment thereof with the
radionuclide under conditions (e.g., pH, salt concentration, and/or
temperature) which facilitate binding of the radionuclide atom or
radionuclide atom-containing molecule or chelate to the antibody or
antigen binding fragment (see, e.g., U.S. Pat. No. 6,001,329).
[0058] Other examples of detectable moieties that can be linked to
an antibody or antigen binding fragment include imaging agents,
such as MRI or computed tomography (CT) contrast agents.
Non-limiting examples of MRI contrast agents include gadolinium and
manganese chelates, iron salts, or gadolinium compounds such as
gadodiamide (OMNISCAN.TM.), gadobenic acid (MULTIHANCE.TM.),
gadopentetic acid (MAGNEVIST.TM.), gadoteridol (PROHANCE.TM.),
gadofosveset (ABLAVAR.TM.) gadoversetamide (OPTIMARK.TM.), or
gadoxetic acid (Eovist, known). Non-limiting examples of CT
contrast agents include iodine based agents such as UROGRAFIN.TM.
TELEBRIX.TM., GASTROGRAFIN.TM., OMNIPAQUE.TM., ULTRAVIST.TM., or
VISIPAQUE.TM. In some embodiments, a nucleic acid encoding an
antibody or an antigen binding fragment such as an scFv fragment is
included in a construct for producing a chimeric immune receptor.
Chimeric immune receptors typically include an extracellular
portion and an intracellular portion, where the extracellular
portion is an antigen binding fragment (e.g., a scFv fragment)
having binding affinity for human FR.beta. and the intracellular
portion is at least the intracellular domain of a signaling
polypeptide such as the CD3 zeta chain, FcR.gamma. chain, or a
kinase such as a Syk cytoplasmic phosphotyrosine kinase. A chimeric
immune receptor further can include at least the intracellular
domain of a costimulatory polypeptide such as the intracellular
domain of CD28 or other costimulatory polypeptide such as OX40
(CD134), CD40L, PD-1, or 4-1BB (CD137). In some embodiments, a
chimeric immune receptor includes an scFv fragment fused to the
nonligand binding part of the extracellular and the entire
transmembrane and intracellular domains of CD28, which is fused
with the intracellular domain of FcR.gamma.. Constructs encoding
chimeric immune receptors can be introduced ex vivo (e.g., using a
retroviral vector) into T cells (e.g., cytotoxic T cells, CD4+ T
cells, or CD8+ T cells) from peripheral lymphocytes of a given
patient, and the resulting engineered T cells containing the
chimeric receptor can be re-introduced into the patient. The
engineered T cells can produce at least one cytokine or lymphokine
(e.g., IL2, IL3, IL4, IL5, IL6, IL9, IL10, IL12, or IFN.gamma.).
Upon binding of the engineered T cells to FR.beta. expressing
target cells, the engineered T cells are activated and can kill the
FR.beta. expressing target cells. See, for example, Eshhar in
"Therapeutic Antibodies. Handbook of Experimental Pharmacology 181"
Y. Chernajovsky, A. Nissim (eds.), 2008; and Pienert et al.,
Immunotherapy, 1(6): 905-912 (2009).
Methods of Using Antibodies
[0059] Typically, a human anti-human FR.beta. mAb or antigen
binding fragment thereof is administered to a mammal such as a
human patient that has been diagnosed with an inflammatory disorder
or a cancer expressing cell surface FR.beta.. In some embodiments,
engineered T cells containing a chimeric immune receptor (see above
section) are administered to the human patient. Non-limiting
examples of inflammatory disorders include atherosclerosis,
ischemia/reperfusion injury, transplantation rejection, vasculitis
such as Wegener's granulomatosus, inflammatory osteoarthritis,
glomerulonephritis, restenosis, systemic sclerosis, fibromyalgia,
sarcoidosis, and autoimmune diseases. Non-limiting examples of
autoimmune diseases include rheumatoid arthritis, systemic lupus
erythematosus (SLE), ulcerative colitis, psoriasis, Type 1 diabetes
(insulin-dependent diabetes mellitus), Crohn's disease, multiple
sclerosis, and Sjogren's disease. Inflammatory disorders also can
include obstructive pulmonary diseases such as asthma or chronic
obstructive pulmonary disease (COPD).
[0060] The antibodies or fragments described herein also can be
administered to a subject suspected of having an inflammatory
disorder. A subject "suspected of having an inflammatory disorder"
is one having one or more signs of the disorder. Signs of such
disorders are well-known to those of skill in the art and include,
without limitation, redness, swelling (e.g., swollen joints), skin
rashes, joint pain, joint pain, loss of joint function, fever,
chills, fatigue, loss of energy, headaches, loss of appetite,
muscle stiffness, insomnia, itchiness, stuffy nose, sneezing,
coughing, or one or more neurologic symptoms such as weakness,
paresthesias, paralysis, dizziness, seizures, or pain. Signs of
diabetes include, without limitation, higher than normal frequency
of urination, unusual thirst, extreme hunger, unusual weight loss,
extreme fatigue, visual problems, and irritability.
[0061] Non-limiting examples of cancers expressing cell surface
FR.beta. include myeloid cancers such as acute myeloid leukemia
(AML) or chronic myeloid leukemia (CML), multiple myeloma, or a
solid cancer containing FR.beta. expressing cells such as squamous
cell carcinoma of the head and neck, or a malignancy of
non-epithelial origin. Treatment of an inflammatory disorder or
cancer can include reducing the severity of the disorder or slowing
progression of the disorder.
[0062] A human anti-human FR.beta. mAb, or antigen binding fragment
thereof, also can be administered prophylactically in subjects at
risk for developing an inflammatory disorder to prevent development
of symptoms of the disorder from occurring, delay onset of
symptoms, or lessen the severity of subsequently developed disorder
symptoms. A subject "at risk of developing an inflammatory
disorder" refers to a subject with a family history of one or more
inflammatory disorders (e.g., a genetic predisposition to one or
more inflammatory disorders) or one exposed to one or more
inflammation-inducing conditions. For example, a subject can have
been exposed to a viral or bacterial superantigen such as, but not
limited to, Staphylococcal enterotoxins (SEs), a Streptococcus
pyogenes exotoxin (SPE), a Staphylococcus aureus toxic
shock-syndrome toxin (TSST-1), a Streptococcal mitogenic exotoxin
(SME) and a Streptococcal superantigen (SSA). An antibody or
fragment thereof also can be administered to deplete activated
macrophages from a human subject. In any case, an amount of the
antibody or antigen binding fragment thereof effective to reduce
the number of FR.beta. positive cells (e.g., macrophages and
monocytes, or cancer cells) in the patient is administered. The
number of FR.beta. positive cells can be determined by doing cell
counts or using a folate-targeted imaging agent.
[0063] Methods described herein can include monitoring the patient
to, for example, determine if the disorder is improving with
treatment. Any method can be used to monitor an inflammatory
disorder or cancer. For example, for rheumatoid arthritis patients,
joint pain and/or stiffness, or bone erosion can be monitored in
the patient. For cancer patients, tumor size, cell count, or cancer
specific markers can be monitored.
[0064] Antibodies or antibody fragments described herein (with or
without linked moieties) may be administered by any available route
including, but not limited to, oral or parenteral routes of
administration such as intravenous, intramuscular, intraperitoneal,
subcutaneous, intrathecal, intraarterial, nasal, transdermal (e.g.,
as a patch), or pulmonary absorption. Antibodies or antibody
fragments may include a delivery agent (e.g., a cationic polymer,
peptide molecular transporter, surfactant, etc.) as a composition
containing a pharmaceutically acceptable carrier. As used herein
the term "pharmaceutically acceptable carrier" includes solvents,
dispersion media, coatings, antibacterial and antifugal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. Supplementary active compounds
can also be incorporated into pharmaceutical formulations as
described herein.
[0065] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Solutions or suspensions
used for parenteral administration can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0066] Pharmaceutical compositions suitable for injectable use
typically include sterile aqueous solutions (where water soluble)
or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. For
intravenous administration, suitable carriers include physiological
saline, bacteriostatic water, the polyoxyl castor oil CREMOPHOR.TM.
EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
In all cases, the composition should be sterile and should be fluid
to the extent that easy syringability exists. Pharmaceutical
formulations are ideally stable under the conditions of manufacture
and storage and should be preserved against the contaminating
action of microorganisms such as bacteria and fungi. In general,
the relevant carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. Prevention of the
action of microorganisms can be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like. In many cases, it
will be advantageous to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride
in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0067] Sterile injectable solutions can be prepared by
incorporating the antibody or antibody fragment in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the purified antibody or antibody fragment into a sterile vehicle
which contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
exemplary methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0068] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the antibody or antibody fragment can be incorporated with
excipients and used in the form of tablets, troches, or capsules,
e.g., gelatin capsules. Oral compositions can also be prepared
using a fluid carrier for use as a mouthwash. Pharmaceutically
compatible binding agents, and/or adjuvant materials can be
included as part of the composition. The tablets, pills, capsules,
troches and the like can contain any of the following ingredients,
or compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose, a disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate or Sterotes;
a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl salicylate, or orange flavoring. Formulations
for oral delivery may advantageously incorporate agents to improve
stability within the gastrointestinal tract and/or to enhance
absorption.
[0069] For administration by inhalation, the antibody or antibody
fragment and a delivery agent are preferably delivered in the form
of an aerosol spray from a pressured container or dispenser which
contains a suitable propellant, e.g., a gas such as carbon dioxide,
or a nebulizer. The present disclosure particularly contemplates
delivery of the compositions using a nasal spray, inhaler, or other
direct delivery to the upper and/or lower airway. According to
certain embodiments, antibody or antibody fragment and a delivery
agent are formulated as large porous particles for aerosol
administration.
[0070] Systemic administration also can be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the purified
polypeptide or protein and delivery agents are formulated into
ointments, salves, gels, or creams as generally known in the
art.
[0071] In certain embodiments, compositions are prepared with
carriers that will protect the antibody or antibody fragment
against rapid elimination from the body, such as a controlled
release formulation, including implants and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be
used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions can also be used as pharmaceutically acceptable
carriers. These can be prepared according to methods known to those
skilled in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0072] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active antibody or antibody fragment calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier.
[0073] The antibody or antibody fragment can be administered at
various intervals and over different periods of time as required.
Those of ordinary skill in the art will appreciate that certain
factors can influence the dosage and timing required to effectively
treat a subject, including but not limited to the severity of the
disease or disorder, previous treatments, the general health and/or
age of the subject, and other diseases present. Generally,
treatment of a subject with an antibody or antibody antigen-binding
fragment as described herein can include a single treatment or, in
many cases, can include a series of treatments. It is furthermore
understood that appropriate doses may depend upon the potency of
the antibody or antibody fragment and may optionally be tailored to
the particular recipient, for example, through administration of
increasing doses until a preselected desired response is achieved.
It is understood that the specific dose level for any particular
animal subject may depend upon a variety of factors including the
activity of the specific polypeptide or protein employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0074] Pharmaceutical formulations as described herein can be
included in a container, pack, or dispenser together with
instructions for administration.
[0075] In some embodiments, an antibody or antigen binding fragment
thereof is administered in combination with one or more
pharmaceutical agents (e.g., an anti-inflammatory agent) or
antibodies.
Articles of Manufacture
[0076] Antibodies or antigen binding fragments described herein
that specifically bind FR.beta. can be combined with packaging
material and sold as a kit for treating inflammatory disorders,
treating a cancer expressing cell surface FR.beta., or depleting
activated macrophages from a subject. The articles of manufacture
may combine one or more human anti-human FR.beta. antibodies or
fragments thereof. In addition, the articles of manufacture may
further include reagents such as secondary antibodies, buffers,
indicator molecules, solid phases (e.g., beads), pharmaceutical
agents, and/or other useful reagents for treating inflammatory
disorders, treating cancer, or depleting activated macrophages from
a subject. Instructions describing how the various reagents are
effective for treating inflammatory disorders, treating cancer, or
depleting activated macrophages from a subject also may be included
in such kits.
[0077] Certain embodiments of methods and compositions provided
herein are further illustrated by the following examples. The
examples are provided for illustrative purposes only, and not to be
construed as limiting the scope or content of the invention in any
way.
EXAMPLES
Example 1
Expression of Recombinant Folate Receptor Beta
[0078] A nucleic acid encoding a fragment of the human folate
receptor beta (FR.beta.) spanning amino acids 22 to 236 (numbering
based on sequence in GenBank Accession No. NP_001107007) was cloned
from pcDNA3 to baculovirus transfer vector pAcGP67 via Sma I and
EcoRI sites. FIG. 1 contains the amino acid sequence (SEQ ID NO:9)
of the FR.beta. protein. Transfer vector pAcGP67 (BD Biosciences
Pharmingen) was co-transfected with BaculoGold.TM. (BD Biosciences
Pharmingen) viral DNA into SF9 insect cells according to the
manufacturer's instruction.
[0079] Recombinant FR.beta. protein produced from pAcGP67 had four
extra residues (ADPG, SEQ ID NO:12) on the N-terminus and five
extra histidine residues on the C-terminus. The recombinant
FR.beta. protein was purified from conditioned medium with a
nickel-chelating column, and further purified with a SUPERDEX.TM.
75 10/300GL gel filtration column in phosphate buffered saline
(PBS). The purified protein was >95% purity as estimated by
SDS-PAGE. The recombinant FR.beta. retained its function of binding
to folate.
Example 2
Antibody Selection by Phage Display
[0080] Purified FR.beta. was used for panning of a human naive (not
deliberately immunized) Fab phage library. Briefly, 2 .mu.g of
FR.beta. was coated on a Maxisorp plate, which was then incubated
with library phage (.about.10.sup.12 pfu) for 2 hr. After
extensively washing the wells with PBS+0.05% TWEEN20.TM.
polysorbate nonionic surfactant (PBST), phage were rescued by
incubating with exponentially growing TG1 bacteria and helper
phage. The panning procedure was repeated three more times with
more stringent conditions in the latter two rounds, including
decreased amounts of FR.beta. protein and additional washes with
PBST. Three hundred colonies were picked from the last two rounds
of panning and rescued with helper phage.
[0081] Two clones were selected and further affinity improved by
light chain shuffling in which two sub-libraries were made by
replacing each light chain sequence with the light chain repertoire
from the original library. The sub-libraries then were further
panned and screened with FR.beta. protein as described above.
[0082] One clone, m923, identified by this method specifically
bound to FR.beta. protein and contained the heavy chain sequence
set forth in SEQ ID NO:7 and the light chain sequence set forth in
SEQ ID NO:8.
[0083] The clone with the highest affinity, m909, was further
characterized as described below. FIGS. 2A and 2B contain the m909
Fab heavy chain sequence (SEQ ID NO:7) and m909 Fab lamba light
chain sequence (SEQ ID NO:17), respectively, with the CDR sequences
underlined, framework region sequences italicized, and constant
region sequence in lowercase, non-italicized text.
[0084] The m909 Fab was expressed in E. coli HB2151 cells. Soluble
Fab was purified from the periplasm using a Ni-chelating column, as
the phagemid carries sequences encoding His6 and FLAG tags at the
C-terminus of the Fab. A single chain form (scFv) of m909 was made
by cloning the VH and VL from the Fab, connected by a linker
(GGGGS; SEQ ID NO:13), into pComb3x. Expression and purification of
scFv were similar to that of the Fab.
[0085] The Fab fragment was converted into an IgG1 molecule by
subcloning the heavy chain variable region and the light chain into
pDR12 to produce pDR12-m909. See, Bender et al., Hum Antibodies
Hybridomas, 4:74-9 (1993). FreeStyle.TM. 293 cells were transfected
with pDR12-m909, and IgG1 was secreted into the medium. IgG1 was
purified with a protein G column. scFv was purified with a
nickel-chelating column. All preparations were dialysed against
PBS.
Example 3
Characterization of the m909 Antibody
[0086] To characterize the m909 antibody, an ELISA was performed as
follows. FR.beta., diluted in PBS, was coated on half of the wells
of a 96 well plate at 50 ng/well overnight at 4.degree. C. Wells
were blocked with 100 .mu.l of 4% milk/PBS (MPBS) for 1 hr at
37.degree. C. For Fab binding kinetics, Fab was titrated from 3000
nM to 0.038 nM (1:5 serial dilutions) then 50 .mu.l of diluted Fab
was added to duplicate wells. After 2 hr incubation at 37.degree.
C., the wells were washed four times with PBST (PBS+0.05%
TWEEN20.TM. polysorbate nonionic surfactant). Bound Fab was
detected by incubating with anti-FLAG-horseradish peroxidase (HRP)
mAb (1:1000) (Sigma) for 1 hr at 37.degree. C. Wells were washed
again with PBST. The HRP substrate
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was
added (50 .mu.l/well), and the absorbance at 405 nm was determined.
For ELISA with IgG, a goat anti-human Fc IgG conjugated with HRP
was used at 1:1000. FIG. 3 is a graph depicting the binding of m909
IgG.sub.1 to FR.beta.. M909 does not detectably bind to FR alpha
(FRI), which shares 70% amino acid identity with FR.beta..
[0087] FR.beta. expression of Chinese hamster ovary (CHO)-K1, CHO
cells stably transfected with human FR.beta. (CHO-FR.beta.), KB
nasopharyngeal epidermoid cells, and preB L1.2 cells was determined
using folate conjugated to fluorescein isothiocyanate (FITC)
(FITC-folate) and mouse mAb 94b. CHO-K1 cells did not detectably
express cell surface FR.beta. while the CHO-FR.beta. cells had high
levels of cell surface FR.beta.. KB and preB L1.2 cells had low but
detectable levels of cell surface FR.beta..
[0088] Binding of m909 to cell surface FR.beta. was determined
using FFC on a FACSCalibur (Beckton Dickinson) flow cytometer.
Aliquots of cells were incubated with primary antibody (m909, or
isotype controls) at the indicated concentrations (see FIGS. 4 and
5) in 250 .mu.l of folate-free RPMI+10% FBS for 1 hr on ice.
Unbound antibodies were washed away with folate-free RPMI.
Secondary antibody goat anti-human IgG conjugated with FITC (Sigma)
was incubated with cells at a concentration of 8 .mu.l/ml for 30
min. m909 Fab and scFv were also pre-incubated with mouse anti-His6
mAb for 30 min at room temperature, before they were added to
cells. For detection of Fab or scFv, 1.6 .mu.g/ml of anti-His6
monoclonal antibody (Qiagen) and 8 .mu.l/ml of goat anti-mouse
IgG-FITC (Sigma) were incubated with cells. Cells were washed and
resuspended in PBS+0.5% BSA for FFC on a FACSCalibur (Beckton
Dickinson) flow cytometer. FIG. 4 shows that in FR.beta.+ cells,
the monovalent format of the m909 antibody (scFv) did not bind to
cell surface FR.beta., whereas the bivalent format, sample 5 (scFv
crosslinked by anti-His6 mAb) shows binding. Fab also does not bind
to FR.beta.. FIG. 5 indicates that in FR.beta.-cells, neither the
monovalent format (scFv) nor bivalent format (IgG) of m909 shows
detectable binding.
[0089] FIGS. 6A and 6B show the concentration-dependent binding of
m909 IgG to FR.beta.+ cells. In FIG. 6A, CHO-FR.beta. cells (a)
were incubated with serial concentrations of control FITC-Isotype
IgG1, and CHO-FR.beta. cells (b), KB cells (c), and CHO-K1 cells
(d) were incubated with serial concentrations of control
FITC-anti-FR.beta.IgG1. m909 bound to FR.beta.-positive cell lines.
FIG. 6B is a graph depicting affinity of FITC-anti-FR.beta.IgG1 for
CHO-FR.beta. cells. Saturation binding fit gave a dissociation
constant (Kd) of 6.393 nM, which is the concentration of free
FITC-anti-FR.beta.IgG1 that would half-saturate FR.beta. on the
cell surface.
Example 4
Cell Lysis by Antibody-Dependent Cell-Mediated Cytotoxicity
[0090] Peripheral blood mononuclear cells (PBMC) were isolated from
healthy human donors with Ficoll-Paque Plus (GE Healthcare).
Collections of blood from donors were approved by NCI-Frederick
Research Donor Program. The viability of isolated cells was
>95%. PBMC were seeded in a 96 well plate in RPMI+10% FBS at
500,000 cells/well. Cells were incubated at 37.degree. C. and
allowed to attach to the plate for 3 hr. Unattached cells were
rinsed off by two washes of warm PBS, cells attached in the wells
were used as the effector cells. Target cells (CHO-K1,
CHO-FR.beta., or preB L1.2 cells) were trypsinized and resuspended
into single cell suspensions. The target cells were incubated with
various concentrations of m909 IgG or control IgG at room
temperature for 30 min then added to effector cells at 10,000
cells/well. The ratio of effector and target cells was 50:1. The
plate was centrifuged at 300 g.times.5 min and incubated at
37.degree. C. for 24 hr. Supernatant (100 .mu.l) was transferred to
an all-white plate and 100 .mu.l of CYTOTOX-ONE.TM. lactate
dehydrogenase detection reagent (Promega) was added to each well.
Lactate dehydrogenase (LDH) released from lysed cells converted the
CytoTox substrate to fluorescent resorufin, which was measured in a
fluorometer at the emission wavelength of 590 nm (excitation
wavelength is 530-560 nm). The percentage of specific lysis was
calculated as the fluorescence of (experimental treatment-effector
cell control)/(high control-target cell control).times.100%.
Fluorescence of target cells alone treated with 1% TRITON X100.TM.
nonionic surfactant was used as high control. Each treatment was
carried out in 6 replicate wells. Each assay plate included control
wells.
[0091] FIGS. 7 and 8 indicate that m909 induced ADCC was correlated
with the level of surface FR.beta. expression. In preB L1.2 cells,
cell surface FR.beta. expression was positive but low, and ADCC was
detectable at approximately 5% (see FIG. 7). In CHO-FR.beta. cells,
expression of surface FR.beta. was much higher, and m909 induced
approximately 20% of cell lysis (see FIG. 8).
Example 5
Species-Specific Binding of Human Anti-Human FR.beta. Monoclonal
Antibody to FR.beta. Positive Cells
[0092] Live Yersinia enterocolitica bacterial cells were injected
intraperitoneally (IP) into BALB/c mice to recruit peritoneal
FR.beta..sup.+ macrophages. Three days after the IP injection,
peritoneal cells were removed and analyzed by flow cytometry using
F4/80 macrophage specific antibody. The cells were stained with
either (a) 100 nM Folate-FITC in the absence (solid black
histogram) or presence of an excess (10 .mu.M) of free folic acid
to competitively occupy FR (filled gray histogram) or (b) 5 nM
FITC-anti-human FR.beta. m909 IgG1 (solid black histogram) or 5 nM
control FITC-Isotype IgG1 (filled gray histogram). The percentage
of FR.beta..sup.+ cells within each gate is shown in FIG. 9A. While
there were approximately 34.23% of FR.beta..sup.+ cells present
(see top panel of FIG. 9A), staining with FITC conjugated
anti-human FR.beta. m909 IgG1 detected only 0.15% of FR.beta..sup.+
cells present.
[0093] Human peripheral blood mononuclear cells (PBMCs) were
stained with folate conjugated to Oregon Green (FOG) and both
phycoerythrin (PE)-anti-CD14 and Tricolor-anti-CD16 antibodies. In
the experiments shown in FIG. 9B, the cells were stained with (a)
100 nM FOG in the absence (solid black histogram) or presence of an
excess (10 .mu.M) of free folic acid to competitively occupy FR
(filled gray histogram) or (b) 5 nM FITC-anti-FR.beta. m909 IgG1
(solid black histogram) or 5 nM control FITC-Isotype IgG1 (filled
gray histogram). The percentage of FR.beta..sup.+ cells within each
gate is shown in FIG. 9B. FITC-anti-FR.beta. m909 IgG1 bound
selectively to CD14.sup.high, CD16.sup.- monocytes.
Other Embodiments
[0094] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
2118PRTArtificial Sequenceheavy chain complementarity determining
region from a human anti-human FR beta antibody 1Gly Tyr Thr Phe
Thr Ser Tyr Ala1 5 27PRTArtificial Sequenceheavy chain
complementarity determining region from a human anti-human FR beta
antibody 2Lys Tyr Ser Gln Lys Phe Gln1 5 312PRTArtificial
Sequenceheavy chain complementarity determining region from a human
anti-human FR beta antibody 3Ala Arg Asp Ile Ser Tyr Gly Ser Phe
Asp Tyr Trp1 5 10 46PRTArtificial Sequencelight chain
complementarity determining region from a human anti-human FR beta
antibody 4Asn Leu Arg Ser Tyr Tyr1 5 53PRTArtificial Sequencelight
chain complementarity determining region from a human anti-human FR
beta antibody 5Gly Lys Asn1 612PRTArtificial Sequencelight chain
complementarity determining region from a human anti-human FR beta
antibody 6His Ser Arg Lys Ser Arg Gly Asn His Leu Leu Phe1 5 10
7229PRTArtificial Sequenceheavy chain sequence from a human
anti-human FR beta antibody 7Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile
Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asp Ile Ser Tyr Gly Ser Phe Asp Tyr Trp Gly Gln
Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn145 150 155 160 Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220 Ser Gly Gln Ala Gly225 8213PRTArtificial Sequencelambda
light chain sequence from a human anti-human FR beta antibody 8Ser
Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10
15 Thr Val Arg Ile Thr Cys Gln Gly Asp Asn Leu Arg Ser Tyr Tyr Ala
20 25 30 Ser Trp Tyr Arg Gln Lys Ser Gly Gln Ala Pro Val Leu Val
Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg
Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile
Thr Ala Ala Gln Ala Glu65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys His
Ser Arg Lys Ser Arg Gly Asn His 85 90 95 Leu Leu Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser
Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn
Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140
Ala Val Thr Val Ala Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val145
150 155 160 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
His Arg Ser Tyr 180 185 190 Ser Cys Gln Val Thr His Glu Gly Ser Thr
Val Glu Lys Thr Val Ala 195 200 205 Pro Thr Glu Cys Ser 210
9255PRTHomo sapiens 9Met Val Trp Lys Trp Met Pro Leu Leu Leu Leu
Leu Val Cys Val Ala1 5 10 15 Thr Met Cys Ser Ala Gln Asp Arg Thr
Asp Leu Leu Asn Val Cys Met 20 25 30 Asp Ala Lys His His Lys Thr
Lys Pro Gly Pro Glu Asp Lys Leu His 35 40 45 Asp Gln Cys Ser Pro
Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr 50 55 60 Ser Gln Glu
Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp65 70 75 80 Asp
His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln 85 90
95 Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln
100 105 110 Gln Val Asn Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp Val
Pro Leu 115 120 125 Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys
His Thr Ser His 130 135 140 Thr Cys Lys Ser Asn Trp His Arg Gly Trp
Asp Trp Thr Ser Gly Val145 150 155 160 Asn Lys Cys Pro Ala Gly Ala
Leu Cys Arg Thr Phe Glu Ser Tyr Phe 165 170 175 Pro Thr Pro Ala Ala
Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys 180 185 190 Val Ser Asn
Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe 195 200 205 Asp
Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala 210 215
220 Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly
Gly225 230 235 240 Leu Leu Leu Ser Leu Ala Leu Met Leu Gln Leu Trp
Leu Leu Gly 245 250 255 10118PRTArtificial Sequenceheavy chain
sequence from a human anti-human FR beta antibody 10Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35
40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Ser Tyr Gly Ser Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
11108PRTArtificial Sequencelambda light chain sequence from a human
anti-human FR beta antibody 11Ser Ser Glu Leu Thr Gln Asp Pro Ala
Val Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile Thr Cys Gln
Gly Asp Asn Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Arg Gln
Lys Ser Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn
Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser
Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Ala Ala Gln Ala Glu65 70 75
80 Asp Glu Ala Asp Tyr Tyr Cys His Ser Arg Lys Ser Arg Gly Asn His
85 90 95 Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
124PRTArtificial SequenceN-terminal amino acids from recombinantly
produced FR beta 12Ala Asp Pro Gly1 135PRTArtificial Sequencelinker
on single chain form of m909 13Gly Gly Gly Gly Ser1 5
146PRTArtificial Sequencelight chain complementarity determining
region from a human anti-human FR beta antibody 14Ser Leu Arg Ser
Asn Tyr1 5 153PRTArtificial Sequencelight chain complementarity
determining region from a human anti-human FR beta antibody 15Gly
Gln Asn1 1612PRTArtificial Sequencelight chain complementarity
determining region from a human anti-human FR beta antibody 16Asp
Ser Arg Val Ser Thr Gly Asn His Val Val Phe1 5 10
17213PRTArtificial Sequencelambda light chain sequence from a human
anti-human FR beta antibody 17Ser Ser Glu Leu Thr Gln Asp Pro Ala
Val Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile Thr Cys Gln
Gly Asp Ser Leu Arg Ser Asn Tyr Ala 20 25 30 Asn Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Gln Asn
Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser
Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Ala65 70 75
80 Asp Glu Ala Asp Tyr Tyr Cys Asp Ser Arg Val Ser Thr Gly Asn His
85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile
Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Lys Ala Asp
Ser Ser Pro Val Lys Ala Gly Val145 150 155 160 Glu Thr Thr Thr Pro
Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu
Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185 190 Ser
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200
205 Pro Thr Glu Cys Ser 210 18108PRTArtificial Sequencelambda light
chain sequence from a human anti-human FR beta antibody 18Ser Ser
Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15
Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Asn Tyr Ala 20
25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile
Tyr 35 40 45 Gly Gln Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe
Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr
Gly Ala Gln Ala Ala65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asp Ser
Arg Val Ser Thr Gly Asn His 85 90 95 Val Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 19688DNAArtificial Sequencenucleotide
sequence encoding m909 heavy chain 19cgaagtgcag ctggtgcagt
ctggggctga ggtgaagaag cctggggcct cagtgaaggt 60ttcctgcaag gcttctggat
acaccttcac tagctatgct atgcattggg tgcgccaggc 120ccccggacaa
aggcttgagt ggatgggatg gatcaacgct ggcaatggta acacaaaata
180ttcacagaag ttccagggca gagtcaccat taccagggac acatccgcga
gcacagccta 240catggagctg agcagcctga gatctgaaga cacggctgtg
tattactgtg cgagagacat 300cagctatggt tcgtttgact actggggcca
gggaaccctg gtcaccgtct cctcagcctc 360caccaagggc ccatcggtct
tccccctggc accctcctcc aagagcacct ctgggggcac 420agcggccctg
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa
480ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt
cctcaggact 540ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc
ttgggcaccc agacctacat 600ctgcaacgtg aatcacaagc ccagcaacac
caaggtggac aagaaagttg agcccaaatc 660ttgtgacaaa actagtggcc aggccggc
68820653DNAArtificial Sequencenucleotide sequence encoding m923
light chain 20gcctcttctg agctgactca ggaccctgct gtgtctgtgg
ccttgggaca gacagtcagg 60atcacatgcc aaggagacaa cctcagaagc tattatgcaa
gctggtaccg gcagaagtca 120ggacaggccc ctgtacttgt catctatggt
aaaaacaacc ggccctcagg gatcccagac 180cgattctctg gctccagctc
aggaaacaca gcttccttga ccatcactgc ggctcaggcg 240gaagatgagg
ctgactatta ctgtcactcc cggaaaagcc gcggtaacca tctgctattc
300ggcggaggga ccaagctgac cgtcctaggt cagcccaagg ctgccccctc
ggtcactctg 360ttcccgccct cctctgagga gcttcaagcc aacaaggcca
cactggtgtg tctcataagt 420gacttctacc cgggagccgt gacagtggcc
tggaaggcag atagcagccc cgtcaaggcg 480ggagtggaga ccaccacacc
ctccaaacaa agcaacaaca agtacgcggc cagcagctat 540ctgagcctga
cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat
600gaagggagca ccgtggagaa gacagtggcc cctacagaat gttcataatt cta
65321662DNAArtificial Sequencenucleotide sequence encoding m909
light chain 21ggcctcttct gagctgactc aggaccctgc tgtgtctgtg
gccttgggac agacagtcag 60gatcacctgc caaggagaca gcctcagaag caactatgca
aactggtacc agcagaagcc 120aggacaggcc cctgtacttg tcatctatgg
tcaaaacaac cggccctcag ggatcccaga 180ccgattctct ggctccagct
caggaaacac agcttccttg accatcactg gggctcaggc 240ggcagatgag
gctgactatt actgtgactc ccgggtcagc actggtaacc atgtggtatt
300cggcggaggg accaagctga ccgtcctagg tcagcccaag gctgccccct
cggtcactct 360gttcccgccc tcctctgagg agcttcaagc caacaaggcc
acactggtgt gtctcataag 420tgacttctac ccgggagccg tgacagtggc
ctggaaggca gatagcagcc ccgtcaaggc 480gggagtggag accaccacac
cctccaaaca aagcaacaac aagtacgcgg ccagcagcta 540tctgagcctg
acgcctgagc agtggaagtc ccacagaagc tacagctgcc aggtcacgca
600tgaagggagc accgtggaga agacagtggc ccctacagaa tgttcataat
tctagataat 660ta 662
* * * * *
References