Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-CXCR4 ANTIBODIES
The present invention relates to monoclonal antibodies against CXCR4 and their
use in treating diseases in which pathogenesis is mediated by CXCR4 and SDF-1.
CXCR4, a chemokine receptor, is a G protein-coupled, seven-transmembrane
receptor. Like other chemokine receptors, CXCR4 plays an important role in
immune
and inflammatory responses by mediating the directional migration and
activation of
leukocytes. CXCR4 is expressed or overexpressed in a variety of cancer cell
lines and
tissues including breast, prostate, lung, ovarian, colon, pancreatic, kidney,
and brain, as
well as non-Hodgkin's lymphoma and chronic lymphocytic leukemia. The only
known
ligand to CXCR4 is stromal cell-derived factor-1 (SDF-1, or CXCL12). The CXCR4
and
SDF-I interaction plays an important role in multiple phases of tumorigenesis,
including
tumor growth, invasion, angiogenesis, and metastasis.
In view of the involvement of CXCR4 in various serious diseases, CXCR4 has
been studied as a therapeutic target. For example, AMD3 100, a bicyclam CXCR4
antagonist, is available for patients with multiple myeloma and non-Hodgkins
lymphoma.
CTCE9908, a peptide CXCR4 antagonist, is currently in Phase lb/11 clinical
trials for
cancer. In addition, antibodies targeting CXCR4 are disclosed in the art (WO
06/089141,
US 07/0059308, and in Carnec et al [Carnec X, Quan L, Olson W, Hazan U, Dragic
T.
(2005) Anti-CXCR4 Monoclonal Antibodies Recognizing Overlapping Epitopes
Differ
Significantly in Their Ability To Inhibit Entry of Human Immunodeficiency
Virus Type
1. Journal of Virology. Feb.2005: 1930-1933]).
Although there are various agents under development that target CXCR4, there
still exists a need for additional therapeutic agents targeting CXCR4. The
antibodies of
the present invention are therapeutically useful CXCR4 antagonists possessing
a number
of desirable properties. Antibodies of the present invention have increased
chemical and
physical stability, and solubility. The present invention provides CXCR4
antibodies that
bind human CXCR4 with high affinity and inhibit human CXCR4 binding to SDF- 1.
High potency permits the use of low doses in therapeutic regimens. In
addition, these
antibodies interfere with the interaction of SDF-1 to CXCR4, and thus reduce
tumorigenesis, including tumor growth, invasion, angiogenesis, and metastasis.
Furthermore, antibodies of the present invention induce apoptosis of tumor
cells.
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The present invention includes a human engineered antibody or a binding
fragment thereof, that binds human CXCR4, and which:
a. inhibits binding of human SDF-la (SEQ ID NO:33) to CXCR4 with an
IC50 for human CXCR4 between 10 nM and 0.05 nM in a human
CXCR4/125I-SDF-la binding inhibition assay as described herein;
b. inhibits migration of cells bearing CXCR4 on their surface with an IC50
between 30 nM and 0.3 nM in the chemotaxis assay as described herein;
and
c. exhibits an affinity, KD between 15 nM and 0.05 nM in the Surface
Plasmon Reasonance (BlAcore) assay as described herein.
The present invention preferably provides a human engineered antibody or a
binding fragment thereof, that binds human CXCR4, and which inhibits binding
of human
SDF-la (SEQ ID NO:33) to CXCR4 with an IC50 for human CXCR4 between 0.5 nM
and 0.05 nM in a human CXCR4/125I-SDF-la binding inhibition assay as described
herein.
The present invention preferably provides a human engineered antibody or a
binding fragment thereof, that binds human CXCR4, and which inhibits migration
of cells
bearing CXCR4 on their surface with an IC50 between 3.0 nM and 0.3 nM in the
chemotaxis assay as described herein.
The present invention preferably provides a human engineered antibody or a
binding fragment thereof, that binds human CXCR4, and which exhibits an
affinity, KD
between 1.0 nM and 0.05 nM in the Surface Plasmon Reasonance (BlAcore) assay
as
described herein.
The present invention preferably provides a human engineered antibody or a
binding fragment thereof, that binds human CXCR4, and which exhibits anti-
tumorigenesis activity by preventing tumor growth in a tumor xenograft model
as
described herein when administered at 1 mg/kg.
The present invention preferably provides a human engineered antibody or a
binding fragment thereof, that binds human CXCR4, and which induces apoptosis
of
tumor cells in an apoptosis assay as described herein when administered
between 2
pg/mL and 10 pg/mL.
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The present invention includes a human engineered antibody or a binding
fragment thereof, that comprises a light chain comprising a light chain
variable region
that comprises framework regions, CDRL1 having the amino acid sequence of SEQ
ID
NO:8, CDRL2 having the amino acid sequence of SEQ ID NO:9, and CDRL3 having
the
amino acid sequence of SEQ ID NO: 10, and a heavy chain comprising a heavy
chain
variable region that comprises framework regions, CDRH1 having the amino acid
sequence of SEQ ID NO:1, CDRH3 having the amino acid sequence of SEQ ID NO:3,
and CDRH2 having an amino sequence selected from the group consisting of SEQ
ID
NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, wherein the
antibody binds to human CXCR4.
Further, the present invention includes an antibody that binds human CXCR4,
wherein a light chain comprises an amino acid sequence of SEQ ID NO:16, and a
heavy
chain comprises an amino acid sequence selected from the group consisting of
SEQ ID
NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.
In addition, the present invention includes an antibody that binds human
CXCR4,
wherein a light chain comprises an amino acid sequence of SEQ ID NO:22, and a
heavy
chain comprises an amino acid sequence selected from the group consisting of
SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.
The present invention also includes an antibody that binds human CXCR4,
wherein an antibody is selected from the group consisting of an antibody
comprising SEQ
ID NO:17 and SEQ ID NO:22, an antibody comprising SEQ ID NO:18 and SEQ ID
NO:22, an antibody comprising SEQ ID NO:19 and SEQ ID NO:22, an antibody
comprising SEQ ID NO:20 and SEQ ID NO:22, and an antibody comprising SEQ ID
NO:21 and SEQ ID NO:22.
The antibodies of the present invention as defined herein are characterized by
having an IC50 of 10 nM or less in a human CXCR4/125I-SDF-la binding
inhibition assay
as described herein. Preferred antibodies of the invention have a binding
affinity for
human CXCR4 of 5.0 nM or less. Most preferred antibodies of the invention have
a
binding affinity for human CXCR4 of 0.5 nM or less. Further preferred,
antibodies of the
present invention have an IC50 for human CXCR4 between 10 nM and 0.05 nM in a
human CXCR4/125I-SDF-la binding inhibition assay as described herein. Further
preferred, antibodies of the present invention have an IC50 for human CXCR4
between
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0.5 nM and 0.05 nM in a human CXCR4/125I-SDF-la binding inhibition assay as
described herein.
The antibodies of the present invention as defined herein are characterized by
having an IC50 of 30 nM or less in a chemotaxis assay as described herein.
Preferred
antibodies of the invention have an IC50 of 15 nM or less in the chemotaxis
assay. More
preferred antibodies of the invention have an IC50 of 3.0 nM or less in the
chemotaxis
assay. Further preferred, antibodies of the present invention have an IC50
between 30 nM
and 0.3 nM in the chemotaxis assay as described herein. Further preferred,
antibodies of
the present invention have an IC50 between 3.0 nM and 0.3 nM in the chemotaxis
assay as
described herein.
The antibodies of the present invention as defined herein are characterized by
having a KD of 15 nM or less in an assay that evaluates the binding activities
of the
antibodies by Surface Plasmon Reasonance (BlAcore) as described herein. More
preferred antibodies of the invention KD of 10 nM or less in the BlAcore
assay. Most
preferred antibodies of the invention have KD of 1.0 nM or less in the BlAcore
assay.
Further preferred, antibodies of the present invention have KD between 15 nM
and 0.05
nM in the BlAcore assay as described herein. Further preferred, antibodies of
the present
invention have KD between 1.0 nM and 0.05 nM in the BlAcore assay as described
herein.
The antibodies of the present invention as defined herein are characterized by
having anti-tumorigenesis activity by preventing tumor growth in a tumor
xenograft
model using NOD/SCID mice and human non-Hodgkin's lymphoma Namalwa cells as
described herein when administered at 10 mg/kg. More preferred antibodies of
the
invention have having anti-tumorigenesis activity by preventing tumor growth
when
administered at 1 mg/kg.
The antibodies of the present invention as defined herein are characterized by
inducing apoptosis of tumor cells in an apoptosis assay as described herein.
More
preferred antibodies of the invention induce nuclear fragmentation and
activation of
caspase 3, hallmarks of apoptosis, in multiple tumor cells including Namalwa
and CEM
cells when administered between 2 pg/mL and 10 pg/mL.
The present invention includes a pharmaceutical composition comprising an
antibody as described herein in combination with one or more pharmaceutically
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acceptable carriers, diluents, or excipients. In addition, the present
invention includes a
pharmaceutical composition for the treatment of tumorigenesis, including tumor
growth,
invasion, angiogenesis, or metastasis, comprising an antibody as variously
described
herein, in combination with one or more pharmaceutically acceptable carriers,
diluents, or
excipients. Further, the present invention includes a pharmaceutical
composition for the
treatment of a cancer selected from the group consisting of breast cancer,
pancreatic
cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's
lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma,
glioblastoma multiforme, and leukemia comprising an antibody as variously
described
herein, in combination with one or more pharmaceutically acceptable carriers,
diluents, or
excipients.
The present invention includes the use of an antibody as described herein for
the
preparation of a medicament for the treatment of tumorigenesis, including
tumor growth,
invasion, angiogenesis, or metastasis. In addition, the present invention
includes the use
of an antibody as described herein for the preparation of a medicament for the
treatment
of a cancer selected from the group consisting of breast cancer, pancreatic
cancer,
melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's
lymphoma,
lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma
multiforme, and leukemia.
The present invention includes a method of treating tumorigenesis, including
tumor growth, invasion, angiogenesis, or metastasis, comprising administering
to a
patient in need of an antibody as described herein. Further, the present
invention includes
a method of treating a cancer selected from the group consisting of breast
cancer,
pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma,
non-
Hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple
myeloma,
glioblastoma multiforme, and leukemia, comprising administering to a patient
in need of
an antibody as described herein.
The general structure of an "antibody," is very well-known in the art. For an
antibody of the IgG type, there are four amino acid chains (two "heavy" chains
and two
"light" chains) that are cross-linked via intra- and inter-chain disulfide
bonds. When
expressed in certain biological systems, antibodies having unmodified human Fc
sequences are glycosylated in the Fc region. Antibodies may be glycosylated at
other
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positions as well. The subunit structures and three-dimensional configurations
of
antibodies are well known in the art. Each heavy chain is comprised of an N-
terminal
heavy chain variable region ("HCVR") and a heavy chain constant region
("HCCR").
The heavy chain constant region is comprised of three domains (CHI, CH2, and
CH3) for
IgG, IgD, and IgA; and 4 domains (CH1, CH2, CH3, and CH4) for IgM and IgE.
Each
light chain is comprised of a light chain variable region (herein "LCVR") and
a light
chain constant region ("LCCR").
The variable regions of each light/heavy chain pair form the antibody binding
site.
The HCVR and LCVR regions can be further subdivided into regions of
hypervariability,
termed complementarity determining regions (CDRs), interspersed with regions
that are
more conserved, termed framework regions (FR). Each HCVR and LCVR are composed
of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus
in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the 3 CDRs of
the
heavy chain are referred to as "CDRH1, CDRH2, and CDRH3" and the 3 CDRs of the
light chain are referred to as "CDRL1, CDRL2 and CDRL3." The CDRs contain most
of
the residues which form specific interactions with the antigen. The assignment
of amino
acids to each domain is in accordance with well-known conventions [e.g.,
Kabat,
"Sequences of Proteins of Immunological Interest," National Institutes of
Health,
Bethesda, Md. (1991)].
Antibodies of the present invention may have a heavy chain constant region
selected from any of the immunoglobulin classes (IgA, IgD, IgG, IgM, and IgE).
Furthermore, antibodies of the present invention contain an Fc portion which
is derived
from human IgG4 Fc region because of its reduced ability to bind complement
factors as
compared to other IgG sub-types.
An antibody may be derived from a single copy or clone, including e.g., any
eukaryotic, prokaryotic, or phage clone. Preferably an antibody of the
invention exists in
a homogeneous or substantially homogeneous population. An antibody can be
intact,
comprising complete or full length constant regions, including the Fc region,
or a portion
or fragment of such an antibody provided that any shortened form comprises the
antigen-
binding portion and retains antigen-binding capability. Such shortened forms
include,
e.g., a Fab fragment, Fab' fragment or F(ab') 2 fragment that includes the
CDRs or the
variable regions of the anti-CXCR4 antibodies disclosed. Furthermore, such
shortened
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antibody forms can be a single chain Fv fragment that may be produced by
joining the
DNA encoding the LCVR and HCVR with a linker sequence. (See, Pluckthun, The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-
Verlag, New York, pp 269-315, 1994). Regardless of whether fragments or
portions are
specified, the term "antibody" as used herein includes such fragments or
portions as well
as single chain forms unless otherwise indicated. As long as the protein
retains the ability
to specifically or preferentially bind CXCR4 and includes a sequence or
sequences
disclosed herein, it is included within the term "antibody." Antibodies of the
invention
can be produced using techniques well known in the art, e.g., recombinant
technologies,
phage display technologies, synthetic technologies or combinations of such
technologies
or other technologies readily known in the art.
The term "human engineered antibody" refers to an antibody having frameworks,
hinge regions, and constant regions of human origin that are identical with or
substantially identical (substantially human) with frameworks and constant
regions
derived from human genomic sequences. Fully human frameworks, hinge regions,
and
constant regions are those human germline sequences as well as sequences with
naturally-
occurring somatic mutations. A human engineered antibody may comprise
framework,
hinge, or constant regions derived from a fully human framework, hinge, or
constant
region containing one or more amino acid substitutions, deletions, or
additions therein.
Often, a human engineered antibody is preferably substantially non-immunogenic
in
humans.
A variety of different human framework sequences may be used singly or in
combination as a basis for the human engineered antibodies of the present
invention.
Preferably, the framework regions of the antibodies of the invention are of
human origin
or substantially human (at least 95%, 97% or 99% of human origin.) The
sequences of
framework regions of human origin may be obtained from The Immunoglobulin
Factsbook, by Marie-Paule Lafranc, Gerard Lefranc, Academic Press 2001, ISBN
012441351.
The framework sequence for the human engineered antibodies of the present
invention serves as the "donor" variable framework region and can be used to
create
additional human engineered antibodies with the same CDRs specified herein
using
methodology known in the art. Furthermore, the framework sequence for the
human
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engineered antibodies of the present invention can be compared to other known
human
framework sequences to generate additional human engineered antibodies. Thus,
this
information can be used to "back-mutate" another selected homologous human
framework region to the human engineered donor amino acid residue at these
positions.
Further, any "rare" amino acids can be detected in additional human frameworks
such
that the concensus or donor human engineered amino acid residue can be used at
the
relevant position.
The term "inhibit" means the ability to substantially antagonize, prohibit,
prevent,
restrain, slow, disrupt, eliminate, stop, reduce or reverse the biological
effects of binding
to the CXCR4 receptor.
"CXCR4" or "human CXCR4" refers to any human CXCR4, as well as
functionally active, mutated forms thereof Examples include, but are not
limited to, SEQ
ID NO:30, SEQ ID NO:31, and SEQ ID NO:32.
A "patient" is a mammal, preferably a human.
The term "treating" (or "treat" or "treatment") means slowing, stopping,
reducing,
or reversing the progression or severity of a symptom, disorder, condition, or
disease.
The term "preventing" (or "prevent" or "prevention") means prohibiting,
restraining, or inhibiting the incidence or occurrence of a symptom, disorder,
condition,
or disease. Acute events and chronic conditions may be treated and prevented.
In an
acute event, antibody is administered at the onset of a symptom, disorder,
condition, or
disease and discontinued when the acute event ends, whereas a chronic symptom,
disorder, condition, or disease is treated over a more protracted time frame.
The term "therapeutically effective amount" refers to the amount or dose of an
antibody of this invention which, upon single or multiple dose administration
to a patient,
provides the desired treatment or prevention. The therapeutically effective
amount can
comprise an amount of about 0.00 1 to 20 mg/kg per single (e.g., bolus),
multiple or
continuous administration.
Particular antibodies of this invention include: an antibody comprising amino
acid
sequences of SEQ ID NOs: 1, 2, 3, 8, 9, and 10; an antibody comprising amino
acid
sequences of SEQ ID NOs: 1, 4, 3, 8, 9, and 10; an antibody comprising amino
acid
sequences of SEQ ID NOs: 1, 5, 3, 8, 9, and 10; an antibody comprising amino
acid
sequences of SEQ ID NOs: 1, 6, 3, 8, 9, and 10; an antibody comprising amino
acid
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sequences of SEQ ID NOs: 1, 7, 3, 8, 9, and 10. The listed sequences represent
CDRH1,
CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively.
Particular antibodies of this invention include: an antibody comprising a LCVR
having an amino acid sequence of SEQ ID NO: 16 and a HCVR having an amino acid
sequence of SEQ ID NO: 11; an antibody comprising a LCVR having an amino acid
sequence of SEQ ID NO: 16 and a HCVR having an amino acid sequence of SEQ ID
NO: 12; an antibody comprising a LCVR having an amino acid sequence of SEQ ID
NO:
16 and a HCVR having an amino acid sequence of SEQ ID NO:13; an antibody
comprising a LCVR having an amino acid sequence of SEQ ID NO: 16 and a HCVR
having an amino acid sequence of SEQ ID NO: 14; an antibody comprising a LCVR
having an amino acid sequence of SEQ ID NO: 16 and a HCVR having an amino acid
sequence of SEQ ID NO: 15.
The present invention includes five antibodies that bind and inhibit CXCR4
activity. In particular, the present invention includes: an antibody
comprising a light
chain having an amino acid sequence of SEQ ID NO: 22 and a heavy chain having
an
amino acid sequence of SEQ ID NO: 17; an antibody comprising a light chain
having an
amino acid sequence of SEQ ID NO: 22 and a heavy chain having an amino acid
sequence of SEQ ID NO: 18; an antibody comprising a light chain having an
amino acid
sequence of SEQ ID NO: 22 and a heavy chain having an amino acid sequence of
SEQ
ID NO: 19; an antibody comprising a light chain having an amino acid sequence
of SEQ
ID NO: 22 and a heavy chain having an amino acid sequence of SEQ ID NO: 20; an
antibody comprising a light chain having an amino acid sequence of SEQ ID NO:
22 and
a heavy chain having an amino acid sequence of SEQ ID NO: 21.
Preferably, an antibody of the present invention wherein all six CDRs, the
HCVR,
the LCVR, the HCVR and the LCVR, the entire heavy chain, the entire light
chain, or the
entire heavy chain and light chain are limited by a particular sequence as
shown by a SEQ
ID NO: herein is further characterized by having an IC50 in a human CXCR4/125I-
SDF-la
binding inhibition assay as described herein of about of 10 nM or less, more
preferably
about 5.0 nM or less, and most preferably of 0.5 nM or less. Further
preferred, an
antibody of the present invention wherein all six CDRs, the HCVR, the LCVR,
the
HCVR and the LCVR, the entire heavy chain, the entire light chain, or the
entire heavy
chain and light chain are limited by a particular sequence as shown by a SEQ
ID NO:
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herein is further characterized by having an IC50 for human CXCR4 between 10
nM and
0.05 nM in a human CXCR4/125I-SDF-la binding inhibition assay as described
herein.
Further preferred, an antibody of the present invention wherein all six CDRs,
the HCVR,
the LCVR, the HCVR and the LCVR, the entire heavy chain, the entire light
chain, or the
entire heavy chain and light chain are limited by a particular sequence as
shown by a SEQ
ID NO: herein is further characterized by having an IC50 for human CXCR4
between 0.5
nM and 0.05 nM in a human CXCR4/125I-SDF-la binding inhibition assay as
described
herein.
More preferably, an antibody of the present invention wherein all six CDRs,
the
HCVR, the LCVR, the HCVR and the LCVR, the entire heavy chain, the entire
light
chain, or the entire heavy chain and light chain are limited by a particular
sequence as
shown by a SEQ ID NO: herein is further characterized by having an IC50 in a
chemotaxis
assay as described herein of about 30 nM or less, more preferably about 15 nM
or less,
and even more preferably 3.0 nM or less. Further preferred, an antibody of the
present
invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the
entire heavy chain, the entire light chain, or the entire heavy chain and
light chain are
limited by a particular sequence as shown by a SEQ ID NO: herein is further
characterized by having an IC50 between 30 nM and 0.3 nM in the chemotaxis
assay as
described herein. Further preferred, an antibody of the present invention
wherein all six
CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the entire heavy chain, the
entire light chain, or the entire heavy chain and light chain are limited by a
particular
sequence as shown by a SEQ ID NO: herein is further characterized by having an
IC50
between 3.0 nM and 0.3 nM in the chemotaxis assay as described herein.
Even more preferably, an antibody of the present invention wherein all six
CDRs,
the HCVR, the LCVR, the HCVR and the LCVR, the entire heavy chain, the entire
light
chain, or the entire heavy chain and light chain are limited by a particular
sequence as
shown by a SEQ ID NO: herein is further characterized by having a KD in an
assay that
evaluates the binding activities of the antibodies by Surface Plasmon
Reasonance
(BlAcore) as described herein of about 15 nM or less, more preferably about 10
nM or
less, and most preferably 1.0 nM or less. Further preferred, an antibody of
the present
invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the
entire heavy chain, the entire light chain, or the entire heavy chain and
light chain are
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limited by a particular sequence as shown by a SEQ ID NO: herein is further
characterized by having a KD in an assay that evaluates the binding activities
of the
antibodies by Surface Plasmon Reasonance (BlAcore) as described herein between
15
nM and 0.05 nM in the BlAcore assay as described herein. Further preferred, an
antibody
of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR
and the
LCVR, the entire heavy chain, the entire light chain, or the entire heavy
chain and light
chain are limited by a particular sequence as shown by a SEQ ID NO: herein is
further
characterized by having a KD in an assay that evaluates the binding activities
of the
antibodies by Surface Plasmon Reasonance (BlAcore) as described herein between
1.0
nM and 0.05 nM in the BlAcore assay as described herein.
More preferably, an antibody of the present invention wherein all six CDRs,
the
HCVR, the LCVR, the HCVR and the LCVR, the entire heavy chain, the entire
light
chain, or the entire heavy chain and light chain are limited by a particular
sequence as
shown by a SEQ ID NO: herein is further characterized by having anti-
tumorigenesis
activity by preventing tumor growth in a tumor xenograft model using NOD/SCID
mice
and human non-Hodgkin's lymphoma Namalwa cells as described herein when
administered at 10 mg/kg, and even more preferably when administered at 1
mg/kg.
Most preferably, an antibody of the present invention wherein all six CDRs,
the
HCVR, the LCVR, the HCVR and the LCVR, the entire heavy chain, the entire
light
chain, or the entire heavy chain and light chain are limited by a particular
sequence as
shown by a SEQ ID NO: herein is further characterized by inducing apoptosis of
tumor
cells in an apoptosis assay as described herein. More preferred, an antibody
of the
present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the
LCVR, the entire heavy chain, the entire light chain, or the entire heavy
chain and light
chain are limited by a particular sequence as shown by a SEQ ID NO: herein is
further
characterized by inducing nuclear fragmentation and activation of caspase 3,
hallmarks of
apoptosis, in multiple tumor cells including Namalwa and CEM cells when
administered
between 2 pg/mL and 10 pg/mL.
EXAMPLES
Antibodies I, II, III, IV, and V can be made and purified as follows. An
appropriate host cell, such as HEK 293 EBNA or CHO, is either transiently or
stably
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transfected with an expression system for secreting antibodies using an
optimal
predetermined HC:LC vector ratio or a single vector system encoding both HC,
such as
SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27
and LC, such as SEQ ID NO: 28. Clarified media, into which the antibody has
been
secreted, is purified using any of many commonly-used techniques. For example,
the
medium may be conveniently applied to a Protein A or G Sepharose FF column
that has
been equilibrated with a compatible buffer, such as phosphate buffered saline
(pH 7.4).
The column is washed to remove nonspecific binding components. The bound
antibody
is eluted, for example, by pH gradient (such as 0.1 M sodium phosphate buffer
pH 6.8 to
0.1 M sodium citrate buffer pH 2.5). Antibody fractions are detected, such as
by SDS-
PAGE, and then are pooled. Further purification is optional, depending on the
intended
use. The antibody may be concentrated and/or sterile filtered using common
techniques.
Soluble aggregate and multimers may be effectively removed by common
techniques,
including size exclusion, hydrophobic interaction, ion exchange, or
hydroxyapatite
chromatography. The purity of the antibody after these chromatography steps is
greater
than 99%. The product may be immediately frozen at -70 C or may be
lyophilized. The
amino acid sequences for these antibodies are provided below.
SEQ ID NOs
Antibody Heavy Light HCVR LCVR
Chain Chain
I 17 22 11 16
II 18 22 12 16
III 19 22 13 16
IV 20 22 14 16
V 21 22 15 16
Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3
I 1 2 3 8 9 10
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II 1 4 3 8 9 10
III 1 5 3 8 9 10
IV 1 6 3 8 9 10
V 1 7 3 8 9 10
Human CXCR4/125I-SDF-1a Binding Inhibition Assay
SDF-1 binding to CXCR4 is the first step in activating the CXCR4 intracellular
signaling pathway. To determine if an antibody can block the interaction of
SDF-1 and
CXCR4, human leukemia CCRF-CEM cells expressing endogenous CXCR4 are used in
an 125I-labeled SDF-la binding assay. The assay is performed in a 96-well U-
bottom,
non-treated polystyrene plate. The binding assay buffer is prepared with RPMI
1640
medium containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(HEPES), pH 7.5, and 0.2% bovine serum albumin (BSA). Briefly, 200 pL reaction
mixtures containing 300 pM ligand (60 pM 1251-SDF-la and 240 pM cold SDF-la),
different concentrations of the test antibody in assay buffer, 100,000 human
CCRF-CEM
cells, and 0.5 mg scintillation proximity assay (SPA) beads are incubated at
room
temperature for 2 hours. Plates are then counted in a liquid scintillation and
luminescence
counter in SPA mode. CXCR4 antagonists decrease the bound radioactivity in
this assay
in a dose-dependent manner. The inhibitory potency (IC50) of a test antibody
is
calculated using GraphPad Prism software, based on the dose-dependent decrease
of
bound radioactivity.
Antibodies exemplified herein exhibit an IC50 value of 10 nM or less in this
assay.
For example, the antibody III exhibits an average IC50 of 0.45 nM in this
assay. The data
demonstrate that antibodies exemplified herein bind to human CXCR4 with high
affinity
and inhibit human CXCR4 binding to SDF- 1.
Chemotaxis Assay
CXCR4/SDF-1 interaction regulates migration (chemotaxis) of cells bearing
CXCR4 on their surface. To determine the antagonist and cellular activities of
a test
antibody, a chemotaxis assay using human histiocytic lymphoma U937 cells that
express
endogenous CXCR4 is employed. Briefly, U937 cells, grown in Dulbecco's
Modified
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Eagle Medium (D-MEM) containing 10% fetal bovine serum, 1% Minimum Essential
Medium (MEM) sodium pyruvate solution, 1% MEM nonessential amino acids, and 1%
L-glutamine, are harvested and washed once with chemotaxis assay buffer (lx
RPMI
medium containing 10 mM HEPES, pH 7.5, and 0.3% BSA.) After washing, cells are
resuspended in assay buffer at a concentration of 5 x 106 cells/mL. The assay
is
performed in a 96-well cell migration plate. Generally, 50 L of cell mixture
with or
without test antibody, ranging from 0.5 pg/mL to 50 pg/mL, is plated on the
upper
chamber, and 30 pL of SDF-la (10 ng/mL) prepared in lx chemotaxis assay buffer
is
added to the lower chamber. After assembly, the plate is incubated for 2.5
hours at 37 C
under 5% carbon dioxide. Following the incubation, 5 pL of cell proliferation
solution is
added into the lower chamber. The plate is then incubated for 60 minutes at 37
C, and the
migrated cells are detected by measuring the absorbance at 492 nm with a
microplate
reader. CXCR4 antagonists inhibit cell migration, reducing the absorbance
reading. The
inhibitory potency (IC50) of a test antibody in this assay is calculated using
GraphPad
Prism software, based on the dose-dependent decrease of absorbance at 492 nm.
Antibodies exemplified herein exhibit an average IC50 value of 30 nM or less
in
this assay. The antibody III exhibits an average IC50 value of 5.90 nM in this
assay. The
data demonstrate that antibodies exemplified herein bind to human CXCR4 with
high
affinity and inhibit human CXCR4 binding to SDF-1.
Evaluation of binding activities of anti-CXCR4 antibodies by Surface Plasmon
Reasonance (BlAcore)
A Biacore 2000 instrument is used to measure binding kinetics and affinity.
The Biacore utilizes the optical properties of surface plasmon resonance to
detect
alteration in protein concentration of interacting molecules within a dextran
biosensor
matrix. Except as noted, all reagents and materials are purchased from Biacore
AB
(Upsala, Sweden). All measurements are performed at 4 C. The binding
experiment is
performed essentially as described in Stenlund et al (Stenlund P, Babcock GJ,
Sodroski J,
Myszka DG. (2003) Capture and reconstitution of G protein-coupled receptors on
a
biosensor surface. Anal Biochem. 316(2):243-50) and Navratilo et al
(Navratilova I,
Sodroski J, Myszka DG. (2005) Solubilization, stabilization, and purification
of
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chemokine receptors using biosensor technology. Anal Biochem. 339(2):271-81).
Running buffer is 50 mM HEPES, 5 mM magnesium chloride, 1 mM calcium chloride,
150 mM sodium chloride, 2 mg/mL BSA, pH 7.5. The human CXCR4 receptor with a C-
terminal linear C9 peptide tag (SEQ ID NO: 29), is cloned and overexpressed in
canine
thymocyte Cf2Th cells in the same manner as described previously by Mirzabekov
et al
(Mirzabekov, N. Bannert, M. Farzan, W. Hofmann, P. Kolchinsky, L. Wu, R. Wyatt
and
J. Sodroski- Enhanced expression, native purification, and characterization of
CCRS, a
principal HIV-1 coreceptor. J. Biol. Chem. 274 (1999), pp. 28745-28750). The C-
terminal linear C9 peptide tag is recognized by the 1D4 monoclonal antibody
(D.D.
Oprian, R.S. Molday, R.J. Kaufman and H.G. Khorana, Expression of a synthetic
bovine
rhodopsin gene in monkey kidney cells. Proc. Natl. Acad. Sci. USA 84 (1987),
pp. 8874-
8878).
Binding is evaluated using multiple analytical cycles as follows. 1D4 Mab
(Monoclonal clone 1D4, University of British Columbia) is immobilized to a CM5
chip
via amine coupling (about 10,000-20,000 Resonance Units antibody). Cells are
resuspended in 20 mM tris(hydroxymethyl)aminomethane (pH 7.0), 0.1 M ammonium
sulphate, 10% glycerol, 5 mM magnesium chloride, 1 mM calcium chloride, plus
complete ethylenediaminetetraacetic acid-free protease inhibitor tablet. 4x106
cells/mL
final for injection onto the chip (i.e. 2.0x106 cells, 0.5 mL final volume)
are used.
Transfected cells and running buffer with detergent (2% cholesteryl
hemisuccinate ester,
10% dodecyl maltoside, 10% 3-[(3-Cholamidopropyl)dimethylammonio]-1-
propanesulfonate) at a ratio of 5:1 (cells to buffer volume ratio) are
transferred into an
auto-mixer. This mixture is incubated for 10 minutes. After incubation 150 L
solubilized receptor is injected over 1D4 surface at a flow rate of 20
pL/minute. The
sample loop is then washed with running buffer. This is followed by an
injection of 20
pL of antibody at flow rate of 100 pL /minute. The chip is then regenerated
with two 10
second pulses of 10 mM sodium hydroxide + 1% n-octyl-(3-D-glucopyranoside at
100
pL/minute. Association rate constants ("koõ") and dissociation rate constants
("koff") for
each cycle are evaluated using a "1:1 with mass transfer" binding model in the
BlAevaluation software. "KD" is the dissociation constant and it is calculated
by the
formula: koff/koõ = KD. The binding parameters are summarized below. The data
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demonstrate that antibodies exemplified herein bind to human CXCR4 with high
affinity
and inhibit human CXCR4 binding to SDF- 1.
Antibody Kon (M-1s-1) Koff (s-1) KD (nM)
I 2.36 x 106 1.61 x 10-3 0.68
II 2.50x106 1.81x103 0.72
III 1.12 x 106 1.72 x 10-3 1.54
IV 7.74 x 105 3.98 x 10-3 5.14
V 1.91x106 1.68x10-3 0.88
Anti-Tumor Activity in a SCID/Namalwa Xenograft Model
SDF-1/CXCR4 interaction appears to play an important role in multiple stages
of
tumorigenesis, including tumor growth, invasion, angiogenesis, and metastasis.
To
evaluate in vivo anti-tumor activity of a test antibody in cancer, a tumor
xenograft model
using NOD/SCID mice and human non-Hodgkin's lymphoma Namalwa cells is
employed. Briefly, 200,000 Namalwa cells mixed with matrigel (1:1) are
implanted
subcutaneously into the rear flank of the animals. The implanted tumor cells
grow as
solid tumors, the dimensions of which can be continuously monitored and
measured using
a caliper. To determine the in vivo efficacy of a test antibody in this model,
animals
(10/group) are treated with different doses of test antibodies dissolved in
saline or
phosphate buffered saline, 48 hours post tumor cell implantation. Antibodies
are dosed
subcutaneously in the range of 1 pg/mouse, 10 pg/mouse, and 100 pg/mouse, and
tumor
volume and body weight are determined every 2 or 3 days. The studies last
generally 3-4
weeks, depending on the tumor growth. The anti-tumor growth activity of a test
antibody
is determined by the percent reduction in tumor volume in treatment groups
compared to
tumor volume in control groups treated with vehicle alone.
Antibody I inhibits tumor growth in this assay when administered at 10
g/mouse,
which is approximately 0.4 mg/kg. The data demonstrate that Antibody I has
tumorigenesis activity by preventing tumor growth.
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SCID/Namalwa Hematological Lymphoma Model
To further investigate anti-tumor activity of CXCR4 antibody in lymphoma, a
hematological lymphoma model is established by injecting 200,000 Namalwa cells
into
SCID mice via tail vein. Generally, the mice injected with tumor cells die in
5-6 weeks.
To test efficacy of the antibody in this model, animals (10 each group) are
treated with 30
pg/mouse or 100 pg/mouse of test antibody 24 hours post tumor cell injection.
The
antibody is dosed subcutaneously once every 4 days for 6 weeks, and the animal
survival
is recorded on daily basis.
Antibody I treatment groups have shown statistically significant survival
benefit
when compared with vehicle and isotype IgG control groups in this
hematological
lymphoma model.
Anti-Tumor Activity in a SCID/CEM Xenograft Model
To evaluate in vivo anti-tumor activity of a test antibody in cancer, a tumor
xenograft model using NOD/SCID mice and CEM cells is employed. Briefly,
5.0x106
CEM cells mixed with matrigel (1:1) are implanted subcutaneously into the rear
flank of
the animals. The implanted tumor cells grow as solid tumors which can be
continuously
monitored and measured by caliber. To determine the efficacy of a test
antibody in this
model, animals (10 each group) are treated with 10 pg/mouse, 30 pg/mouse, or
100
pg/mouse of test antibody 24 hours post tumor cell implantation. The antibody
is dosed
subcutaneously once every 4 days, and the tumor volume and body weight are
measured
every 2 or 3 days.
A dose-dependent tumor growth inhibition is observed among Antibody I
treatment groups compared with vehicle and isotype IgG control groups.
Antibody I at
all three doses inhibits tumor growth significantly.
Apoptosis Assay
To investigate if CXCR4 antibodies induce apoptosis, multiple tumor cell lines
expressing high levels of CXCR4 are treated with test antibody. The cells are
treated with
different concentrations of test antibody for 2-4 days in their growth medium
with 1% or
10 % FBS. After treatment, cells are fixed with 3.7% formaldehyde and washed
in D-
PBS. Cells are permeabilized with 0.1% Triton X-100 in D-PBS, washed and
blocked in
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D-PBS containing 1% BSA. Cells are then incubated for 1 hour with rabbit anti-
activated
Caspase3 polyclonal antibody (Cat# 557135 BD Biosciences, NC) diluted in D-PBS
with
1% BSA. Cells are washed 2 times with D-PBS then incubated for 1 hour with
Alexa
Fluor 488 goat anti Rabbit IgG (Invitrogen, Carlsbad, CA) and 200ng/mL Hoechst
33342
(Invitrogen, Carlsbad, CA) diluted in D-PBS with 1% BSA. Stained plates are
scanned
using ArrayScan Vti (Cellomics, Pittsburgh, Pennsylvania) and the Target
Activation
bioapplication is used for quantitation of fluorescent signal.
The results demonstrate that Antibody I induces nuclear fragmentation and
activation of caspase 3 in multiple tumor cells including Namalwa and CEM
cells.
Nuclear fragmentation and caspase 3 activation are hallmarks of apoptosis.
Therefore, the
data demonstrate that Antibody I induces apoptosis of tumor cells when
administered
between 2 pg/mL and 10 pg/mL.
To further confirm that Antibody I induces apoptosis, annexin V changes are
investigated by flow cytometry in Namalwa cells after treatment with test
antibody or
isotype IgG control. Antibody I induces a dose-dependent increase of annexin
V, while
isotype IgG has no effect. Furthermore, Antibody I induces apoptosis which is
also
observed in CEM xenograft tumors by TUNEL staining.
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SEQ ID Listing
Heavy Chain CDRs
SEQ ID NO:1 GFTSTDYYFS
SEQ ID NO:2 FIRTKSKGYTTEYSGSVKG
SEQ ID NO:3 EPITTDPRDY
SEQ ID NO:4 FIRSKSKGYTTEYSGSVKG
SEQ ID NO:5 FIRYKSKGYTTEYSGSVKG
SEQ ID NO:6 FIRNKRKGYTTEYSGSVKG
SEQ ID NO:7 FIRHKSKGYTTEYSGSVKG
Light Chain CDRs
SEQ ID NO:8 KSSQSLFNSRTRKKYLA
SEQ ID NO:9 WASKRKS
SEQ ID NO:10 KQSRFLRA
Heavy Chain Variable Regions
SEQ ID NO:11
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRTK
SKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSS
SEQ ID NO:12
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRSK
SKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSS
SEQ ID NO:13
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRYK
SKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSS
SEQ ID NO:14
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRNK
RKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSS
SEQ ID NO:15
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRHK
SKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSS
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Light Chain Variable Regions
SEQ ID NO:16
DIVMTQSPDSLAVSLGERATINCKSSQSLFNSRTRKKYLAWYQQKPGQPPKLLIY
WASKRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSRFLRAFGQGTKLE
IK
Complete Heavy Chains
SEQ ID NO:17
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRTK
SKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLG
SEQ ID NO:18
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRSK
SKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLG
SEQ ID NO:19
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRYK
SKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLG
SEQ ID NO:20
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRNK
RKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
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GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLG
SEQ ID NO:21
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRHK
SKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRD
YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLG
Complete Light Chain
SEQ ID NO:22
DIVMTQSPDSLAVSLGERATINCKSSQSLFNSRTRKKYLAWYQQKPGQPPKLLIY
WASKRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSRFLRAFGQGTKLE
IKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKV QWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Nucleotide Sequences - Heavy Chain Variable Region
SEQ ID NO:23
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGA
CGAAGTCGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGAT
TCACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAG
CCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACC
ACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
SEQ ID NO:24
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGT
CTAAGTCGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGATT
CACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAGC
CTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACCA
CCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
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SEQ ID NO:25
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGT
ATAAGTCGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGATT
CACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAGC
CTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACCA
CCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
SEQ ID NO:26
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGA
ACAAGCGGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGAT
TCACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAG
CCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACC
ACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
SEQ ID NO:27
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGC
ACAAGTCGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGAT
TCACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAG
CCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACC
ACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
Nucleotide Sequences - Light Chain Variable Region
SEQ ID NO:28
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAG
GGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGTTCAACAGCCGGACCCGG
AAGAAGTACCTGGCCTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGC
TCATTTACTGGGCCAGCAAGAGAAAGAGCGGGGTCCCTGACCGATTCAGTGG
CAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAA
GATGTGGCAGTTTATTACTGTAAGCAGAGCCGTTTTCTGAGAGCCTTTGGCCA
AGGGACCAAGCTGGAGATCAAA
C-Terminal Linear C9 Peptide Tag for Human CXCR4 Receptor
SEQ ID NO:29 TETSQVAPA
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Human CXCR4
SEQ ID NO: 30
MEGISSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFL
TGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWY
FGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVG
V WIPALLLTIPDFIFANV SEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVIL
SCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEF
ENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKG
KRGGHSSVSTESESSSFHSS
Human CXCR4 isoform A
SEQ ID NO:31
MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIV
GNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNF
LCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIP
ALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYC
IIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTV
HKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGG
HSSVSTESESSSFHSS
Human CXCR4 isoform B
SEQ ID NO: 32
MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNG
LVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCK
AVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALL
LTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIIS
KLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHK
WISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSS
VSTESESSSFHSS
Human SDF-1 a
SEQ ID NO:33
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKL
KWIQEYLEKALNK
