U.S. patent application number 16/590152 was filed with the patent office on 2020-07-23 for treatment of hematologic malignancies with an anti-cxcr4 antibody.
The applicant listed for this patent is BRISTOL-MYERS SQUIBB COMPANY. Invention is credited to Josephine M. Cardarelli, MICHELLE R. KUHNE, Chin Pan.
Application Number | 20200231683 16/590152 |
Document ID | / |
Family ID | 47179025 |
Filed Date | 2020-07-23 |
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United States Patent
Application |
20200231683 |
Kind Code |
A1 |
KUHNE; MICHELLE R. ; et
al. |
July 23, 2020 |
TREATMENT OF HEMATOLOGIC MALIGNANCIES WITH AN ANTI-CXCR4
ANTIBODY
Abstract
The present disclosure provides human monoclonal antibodies that
bind specifically to CXCR4 with high affinity. This disclosure also
provides a method for treating a subject afflicted with a
CXCR4-expressing cancer, in particular a hematological malignancy
such as multiple myeloma, acute myeloid leukemia, or non-Hodgkin's
lymphoma, comprising administering to the subject a therapeutically
effective amount of a pharmaceutical composition comprising an
anti-CXCR4 antibody of the disclosure. The disclosure further
provides a kit for treating a cancer in a subject comprising a dose
of an anti-CXCR4 antibody and instructions for using the anti-CXCR4
antibody in the therapeutic methods of the disclosure.
Inventors: |
KUHNE; MICHELLE R.; (San
Francisco, CA) ; Pan; Chin; (Los Altos, CA) ;
Cardarelli; Josephine M.; (San Carlos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRISTOL-MYERS SQUIBB COMPANY |
Princeton |
NJ |
US |
|
|
Family ID: |
47179025 |
Appl. No.: |
16/590152 |
Filed: |
October 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14356996 |
May 8, 2014 |
10428151 |
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PCT/US2012/064395 |
Nov 9, 2012 |
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16590152 |
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61557815 |
Nov 9, 2011 |
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61569113 |
Dec 9, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/675 20130101;
A61K 31/454 20130101; A61K 31/573 20130101; C07K 2317/73 20130101;
A61P 35/00 20180101; C07K 2317/21 20130101; A61K 31/136 20130101;
A61P 43/00 20180101; A61K 39/39558 20130101; A61K 31/69 20130101;
A61K 31/4184 20130101; C07K 2317/76 20130101; A61K 45/06 20130101;
C07K 16/2866 20130101; A61K 31/7068 20130101; A61K 2039/505
20130101; A61P 35/02 20180101; A61K 31/7048 20130101; A61P 35/04
20180101; A61K 31/555 20130101; A61K 31/573 20130101; A61K 2300/00
20130101; A61K 31/69 20130101; A61K 2300/00 20130101; A61K 31/454
20130101; A61K 2300/00 20130101; A61K 31/136 20130101; A61K 2300/00
20130101; A61K 31/7048 20130101; A61K 2300/00 20130101; A61K
31/7068 20130101; A61K 2300/00 20130101; A61K 31/4184 20130101;
A61K 2300/00 20130101; A61K 31/675 20130101; A61K 2300/00 20130101;
A61K 31/555 20130101; A61K 2300/00 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06; A61K 31/136 20060101
A61K031/136; A61K 31/4184 20060101 A61K031/4184; A61K 31/454
20060101 A61K031/454; A61K 31/555 20060101 A61K031/555; A61K 31/573
20060101 A61K031/573; A61K 31/675 20060101 A61K031/675; A61K 31/69
20060101 A61K031/69; A61K 31/7048 20060101 A61K031/7048; A61K
31/7068 20060101 A61K031/7068; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for inhibiting growth and/or inducing apoptosis of
multiple myeloma cells in a subject afflicted with multiple
myeloma, said method comprising administering to the subject a
therapeutically effective amount of an antibody or an
antigen-binding fragment thereof which specifically binds to a
human CXCR4 receptor.
2. The method of claim 1, wherein the multiple myeloma is relapsed
or refractory multiple myeloma.
3. The method of claim 1, wherein the antibody or fragment thereof
is administered to the subject as monotherapy.
4. The method of claim 1, wherein the antibody or fragment thereof
is administered to the subject in combination with surgery,
radiation and/or one or more therapeutic agents.
5. The method of claim 1, wherein the antibody or fragment thereof
inhibits the activity of the CXCR4 receptor and increases
sensitivity of the multiple myeloma cell to a chemotherapeutic
agent.
6. The method of claim 1, further comprising administering at least
one chemotherapeutic agent in combination with the antibody or
fragment thereof.
7. The method of claim 6, wherein the at least one chemotherapeutic
agent is: (a) lenalidomide and dexamethasone; or (b) bortezomib and
dexamethasone.
8. The method of claim 1, wherein the antibody or fragment thereof
is a chimeric, humanized, or human antibody or a fragment
thereof.
9. The method of claim 1, wherein the antibody or fragment thereof
is an IgG1 or IgG4 antibody or a fragment thereof.
10. The method of claim 3, wherein the antibody or fragment thereof
is administered to the subject at a dose ranging from about 0.1 to
10 mg/kg body weight.
11. The method of claim 10, wherein the dose is 0.3, 1, 3 or 10
mg/kg body weight.
12. The method of claim 7, wherein the CXCR4 antibody or fragment
thereof is administered to the subject in combination with
lenalidomide and dexamethasone in a dosage regimen comprising: (a)
the CXCR4 antibody or fragment (1, 3, or 10 mg/kg) administered as
an intravenous infusion on Days 1, 8, 15, 22, 29 and 36 (Cycle 1)
and on Days 1, 8, 15, and 22 (Cycle 2 and subsequent cycles); (b)
lenalidomide (25 mg) orally administered for 21 days (Days 15-35;
Cycle 1) and Days 1-21 (Cycle 2 and subsequent cycles); and (c)
dexamethasone (40 mg) administered on Days 15, 22, 29, and 36
(Cycle 1) and on Days 1, 8, 15, and 22 (Cycle 2 and subsequent
cycles).
13. The method of claim 7, wherein the CXCR4 antibody or fragment
thereof is administered to the subject in combination with
bortezomib and dexamethasone in a dosage regimen comprising: (a)
the CXCR4 antibody or fragment (1, 3, or 10 mg/kg) administered as
an intravenous infusion on Days 1, 8, 15, 22 and 29 (Cycle 1) and
on Days 1, 8 and 15 (Cycle 2 and subsequent cycles); (b) bortezomib
(1.3 mg/m.sup.2) administered as an intravenous push on Days 15,
18, 22, and 25 (Cycle 1) and on Days 1, 4, 8, 11 (Cycle 2 and
subsequent cycles); and (c) dexamethasone (20 mg) administered on
Days 15, 16, 18, 19, 22, 23, 25 and 26 (Cycle 1) and on Days 1, 2,
4, 5, 8, 9, 11 and 12 (Cycle 2 and subsequent cycles).
14. The method of claim 1, wherein the antibody or fragment thereof
is a monoclonal antibody or a fragment thereof.
15. The method of claim 1, wherein the subject is a human.
16. A kit comprising a dose of an anti-CXCR4 antibody or fragment
thereof that specifically binds to a CXCR4 receptor for use in a
method of inhibiting growth and/or inducing apoptosis of multiple
myeloma cells in a subject afflicted with multiple myeloma and
thereby treating the subject, wherein the CXCR4 receptor is
expressed on the surface of the multiple myeloma cells in the
subject and wherein the method comprises administering to the
subject the antibody or fragment thereof for directly inhibiting
growth and/or inducing apoptosis of the multiple myeloma cells in
the subject, and wherein the kit further comprises instructions for
using the anti-CXCR4 antibody according to claim 1.
17. The kit for use according to claim 16, wherein the anti-CXCR4
antibody or fragment thereof comprises comprises a heavy chain
variable region comprising CDR1, CDR2 and CDR3 domains comprising
consecutively linked amino acids having the sequences set forth in
SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9, respectively, and a
light chain variable region comprising CDR1, CDR2 and CDR3 domains
comprising consecutively linked amino acids having the sequences
set forth in SEQ ID NO: 13, SEQ ID NO: 17 and SEQ ID NO: 21,
respectively.
18. The kit for use according to claim 16, wherein the anti-CXCR4
antibody or fragment thereof comprises a heavy chain variable
region comprising consecutively linked amino acids having the
sequence set forth in SEQ ID NO: 25, and a light chain variable
region comprising consecutively linked amino acids having the
sequence set forth in SEQ ID NO: 29.
19. The kit for use according to claim 16, wherein the anti-CXCR4
antibody is BMS-936564.
20. The kit of claim 15, which further comprises at least one
chemotherapeutic agent for use in combination with the anti-CXCR4
antibody or fragment thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
14/356,996, filed May 8, 2014, which is the 35 U.S.C. .sctn. 371
national stage application of PCT International Application No.
PCT/US2012/064395, filed Nov. 9, 2012, which claims the benefit of
U.S. Provisional Application Nos. 61/569,113, filed Dec. 9, 2011,
and 61/557,815, filed Nov. 9, 2011, the entire contents of each of
which are hereby incorporated into this application by
reference.
[0002] Throughout this application, various publications are
referenced in parentheses by author name and date, or by Patent No.
or Publication No. Full citations for these publications may be
found at the end of the specification immediately preceding the
claims. The disclosures of these publications in their entireties
are hereby incorporated by reference into this application in order
to more fully describe the state of the art as known to those
skilled therein as of the date of the invention described and
claimed herein. However, the citation of a reference herein should
not be construed as an acknowledgement that such reference is prior
art to the present invention.
FIELD OF THE INVENTION
[0003] The present disclosure relates to human monoclonal
antibodies that bind specifically to native human CXCR4 expressed
on a cell surface, and the use of these antibodies in methods of
treating cancer, particularly hematologic malignancy, including
acute myeloid leukemia (AML), multiple myeloma (MM), and
non-Hodgkin's lymphomas (NHLs) such as chronic lymphoid leukemia
(CLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma
(DLBCL).
BACKGROUND OF THE INVENTION
[0004] Chemokines are a family of about 50 small proteins that
modulate cell trafficking and angiogenesis and also play a
significant role in the tumor microenvironment (Vicari et al.,
2002). Depending on their structure, chemokines are classified as
C-C chemokines (containing a cysteine-cysteine motif) or C-X-C
chemokines (containing a cysteine-X-cysteine motif). Receptors that
bind such chemokines thus are classified as members of the CCR
family or CXCR family, respectively.
[0005] One member of the CXCR family is the CXCR4 receptor (CXCR4),
also known as CD184, a seven-transmembrane domain G-protein coupled
receptor consisting of an extra-cellular N-terminal tail and three
extra-cellular loops. The intracellular carboxy terminus of CXCR4
is coupled to a heterotrimeric G-protein consisting of .beta. and
.gamma. subunits and a pertussis toxin-sensitive Gi .alpha. subunit
(Loetscher et al., 1994). To date, only one ligand for CXCR4, a
chemokine known as CXCL12 (also known, and used interchangeably
herein, as stromal cell-derived factor-1 or SDF-1) has been
identified (Bleul et al., 1996; Oberlin et al., 1996). CXCL12
binding to CXCR4 stimulates activation of phospholipase C and
subsequently results in an elevation of cytosolic free calcium.
Ligation of CXCR4 ultimately leads to induction of chemotaxis and
migration (Tachibana et al., 1998; Zou et al., 1998). CXCR4 also
plays a role in embryogenesis, homeostasis and inflammation.
Studies with mice engineered to be deficient in CXCR4 or CXCL12
implicate the CXCR4/CXCL12 pathway in organ vascularization, as
well as in the immune and hematopoietic systems (Tachibana et al.,
1998). Further, CXCR4 has been shown to function as a coreceptor
for T lymphotrophic HIV-1 isolates (Feng et al., 1996).
[0006] In healthy adults, CXCR4 is predominantly expressed on
hematopoietic lineage cells including B and T cells, monocytes,
macrophages, NK, and dendritic cells, as well as CD34.sup.+ bone
marrow (BM) progenitor cells (Lee et al., 1999). Low levels of
CXCR4 are also expressed on endothelial and epithelial cells,
astrocytes, and neurons (Gupta et al., 1998; Hesselgesser et al.,
1997). CXCL12 has been shown to induce endothelial cell migration
and proliferation and, together with VEGF, has been shown to
enhance neoangiogenesis (Guleng et al., 2005). Over-expression of
CXCR4 has also been found in 75% of cancers including leukemias,
lymphomas, pancreatic, breast, ovarian, lung, prostate and
colorectal tumors, and the interaction between CXCL12 and is
essential for homing and maintaining hematopoietic stem cells
within the BM microenvironment (Mohle et al., 1998). Plerixafor
(AMD3100; Mozobil), a bicyclam antagonist of CXCR4, has been shown
to mobilize stem cells into the bloodstream (Dar et al., 2011).
AMD3100 and AMD3465, another CXCR4 antagonist bicyclam, increase
chemosensitization of AML tumor cells by blocking CXCR4/CXCL12
signaling (Nervi et al., 2009; Zeng et al., 2009).
[0007] AML is a fast-growing cancer of the myeloid line of blood
cells, characterized by the rapid growth of abnormal white blood
cells that accumulate in the BM and interfere with the production
of normal blood cells. In AML, CXCR4 is highly expressed on the
CD34.sup.+ fraction of BM cells. Lower levels of CXCR4 on AML cells
correlate with a better prognosis resulting in a longer relapse
free and overall survival. The lower CXCR4 receptor expression
attenuates migration of primary AML cells toward CXCL12 expressed
in the chemo-protected environment of the BM (Tavor et al.,
2004).
[0008] Multiple myeloma (MM) is a form of cancer that results from
the malignant proliferation of plasma cells. After non-Hodgkin's
lymphoma, it is the second most frequent hematological cancer, with
approximately 80,000 new cases worldwide (20,000 in the United
States), and approximately 62,000 deaths per year (10,500
deaths/year in the U.S.) (Jemal et al., 2008; 2009). MM cells grow
preferentially in the BM where they interfere with the production
of normal blood cells and normal antibodies, resulting in
immunodeficiency, skeletal destruction, hypocalcaemia, BM and renal
failure. In addition to AML, serum levels of CXCL12 are elevated in
patients with MM, and CXCR4 expression increases in extramedullary
plasmacytoma, a manifestation of an advanced stage of MM.
Furthermore, blockade of the CXCL12/CXCR4 axis attenuates migration
of MM cells and homing of these cells to the BM (Alsayed et al.,
2007).
[0009] Non-Hodgkin lymphomas include any of a diverse group of
cancers of lymphocytes other than Hodgkin's lymphomas. NHLs can
occur at any age and are often marked by lymph nodes that are
larger than normal, fever, and weight loss. The many different
types of NHL vary significantly in their severity, from very
aggressive (fast-growing) to indolent (slow-growing) types, and
they can be formed from either B-cells or T-cells. B-cell NHLs
include Burkitt's lymphoma, chronic lymphocytic leukemia/small
lymphoid lymphoma (CLL/SLL), diffuse large B-cell lymphoma (DLBCL),
follicular lymphoma (FL), immunoblastic large cell lymphoma,
precursor B-lymphoblastic lymphoma, and mantle cell lymphoma.
T-cell NHLs include mycosis fungoides, anaplastic large cell
lymphoma, and precursor T-lymphoblastic lymphoma. It is estimated
that there will be approximately 70,000 new cases of NHLs in the
United States in 2012, which will result in about 19,000 deaths.
High-level CXCR4 expression has been demonstrated in 18 out of 19
primary NHL cell lines tested (Bertolini et al., 2002). It has also
been shown that CXCL12 enhances migration of follicular NHL cells
(Corcione et al., 2000), and the CXCR4-CXCL12 circuitry appears to
be crucial for migration of CLL cells (Burger et al., 1999).
[0010] Human anti-CXCR4 monoclonal antibodies that exhibit numerous
desirable properties have previously been described in PCT
International Publication No. WO 2008/060367 (Application No.
PCT/US2007/021152), claiming priority to U.S. Provisional
Application No. 60/827,851, filed Oct. 2, 2006. The disclosures of
both these applications are hereby incorporated in their entireties
by reference into this application. As disclosed in WO 2008/060367,
in vitro studies demonstrate that these monoclonal antibodies bind
to CXCR4-expressing cells with low nanomolar affinity, block CXCL12
binding to CXCR4-expressing cells, and inhibit CXCL12-induced
migration and calcium flux with low nanomolar EC.sub.50 values. One
of the fully human monoclonal antibodies, BMS-936564, (designated
F7 in WO 2008/060367, and also previously designated MDX-1338, all
three designations being used interchangeably herein), which
exhibited unexpectedly advantageous anti-solid tumor properties in
preclinical studies, has been selected for further investigation to
determine its activity against hematologic cancers in vivo and to
further elucidate the mechanisms underlying its anti-cancer
activity. The BMS-936564 antibody has also entered Phase I clinical
studies in patients with relapsed/refractory AML, MM, and NHLs.
SUMMARY OF THE INVENTION
[0011] The present disclosure provides isolated monoclonal
antibodies, in particular human monoclonal antibodies, that bind to
human CXCR4 and that exhibit numerous properties that are desirable
in a therapeutic antibody. These properties include the ability to
bind with low nM affinity to native human CXCR4 expressed on a cell
surface, inhibit SDF-1 binding to human CXCR4 with an EC.sub.50 for
inhibition of 50 nM or less, inhibit SDF-1-induced calcium flux in
cells expressing CXCR4 with an EC.sub.50 for inhibition of 3 nM or
less, inhibit SDF-1-induced migration of cells expressing CXCR4
with an EC.sub.50 for inhibition of 50 nM or less, inhibit
capillary tube formation by human umbilical vein endothelial cells
(HuVECs), induce apoptosis in a wide variety of cells expressing
CXCR4, inhibit tumor cell proliferation in vitro, inhibit tumor
growth in vivo, inhibit metastases of CXCR4.sup.+ tumor cells
and/or increase survival time of a CXCR4.sup.+ tumor-bearing
subject.
[0012] In a preferred aspect, this disclosure pertains to isolated
monoclonal antibody, preferably a human monoclonal antibody, or an
antigen-binding portion thereof, wherein the monoclonal antibody:
[0013] (a) binds to native human CXCR4 expressed on a cell surface;
[0014] (b) inhibits binding of SDF-1 (CXCL12) to human CXCR4;
[0015] (c) inhibits SDF-1-induced calcium flux in cells expressing
human CXCR4; [0016] (d) inhibits SDF-1-induced migration of cells
expressing human CXCR4; and [0017] (e) inhibits capillary tube
formation by human umbilical vein endothelial cells. Even more
preferably, the antibody also induces apoptosis of cells expressing
human CXCR4, induces tumor cell apoptosis in vivo, and/or inhibits
growth of CXCR4.sup.+ tumor cells.
[0018] This disclosure also provides a method for treating a
subject afflicted with a CXCR4-expressing cancer, including a
hematologic malignancy, comprising administering to the subject a
therapeutically effective amount of an anti-CXCR4 antibody that
specifically binds to human CXCR4 expressed on a cell surface. In
certain embodiments, the anti-CXCR4 antibody inhibits the activity
of CXCR4. In preferred embodiments, the anti-CXCR4 antibody induces
apoptosis of CXCR4-expressing target cells. Accordingly, the
anti-CXCR4 antibody is used in certain embodiments as monotherapy.
In other embodiments, the anti-CXCR4 antibody is used in
combination with other anti-cancer agents. In preferred
embodiments, the hematologic malignancy is MM, AML, or NHLs. In
preferred embodiments, the antibody is a human antibody. More
preferably, the antibody is BMS-936564.
[0019] The disclosure further provides a use of a CXCR4 antibody
for the preparation of a pharmaceutical composition for treating a
subject afflicted with a cancer, including a hematologic
malignancy.
[0020] This disclosure also provides a kit for treating a cancer in
a subject, the kit comprising: (a) a dose of an anti-CXCR4
antibody; and (b) instructions for using the anti-CXCR4 antibody in
any of the methods described herein. In a preferred embodiment, the
anti-CXCR4 antibody is BMS-936564.
[0021] Other features and advantages of the instant disclosure will
be apparent from the following detailed description and examples,
which should not be construed as limiting. The contents of all
references, GENBANK.RTM. entries, patents and published patent
applications cited throughout this application are expressly
incorporated herein by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIGS. 1A and 1B: FIG. 1A shows the nucleotide sequence (SEQ
ID NO: 33) and amino acid sequence (SEQ ID NO: 25) of the heavy
chain variable region of the F7 (BMS-936564) human monoclonal
antibody. The CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 5) and CDR3
(SEQ ID NO: 9) regions are delineated and the V, D and J germline
derivations are indicated. FIG. 1B shows the nucleotide sequence
(SEQ ID NO: 37) and amino acid sequence (SEQ ID NO: 29) of the
light chain variable region of F7. The CDR1 (SEQ ID NO: 13), CDR2
(SEQ ID NO: 17) and CDR3 (SEQ ID NO: 21) regions are delineated and
the V and J germline derivations are indicated.
[0023] FIG. 2 shows the binding of human anti-CXCR4 antibodies F7,
F9, D1 and E2 to CEM cells that express native human CXCR4 on the
cell surface.
[0024] FIG. 3 shows antibody competition for binding to CEM cells
between FITC-labeled anti-CXCR4 antibody F9 and a panel of
unlabeled human anti-CXCR4 antibodies.
[0025] FIGS. 4A-4D show a flow cytometric analysis of BMS-936564
binding. The antibody binds to AML cell lines Nomo-1 and HL-60
(FIG. 4A), CXCR4-transfected R1610, CEM and Ramos cell lines (FIG.
4B), MM cell lines, JJN-3R, and MOLP8 (FIG. 4C), and primary AML
patient blood cells (FIG. 4D).
[0026] FIG. 5 shows inhibition of binding of .sup.125I-labeled
CXCL12 to CXCR4 expressed on CEM cells by anti-CXCR4 human
antibodies F7 (BMS-936564), F9 and D1. The E2 antibody does not
inhibit binding of CXCL12 to CEM cells.
[0027] FIGS. 6A-6C: FIGS. 6A-6C show inhibition of binding of
.sup.125I-labeled CXCL12 to CEM cells by anti-CXCR4 antibody
MDX-1338 (BMS-936564) (FIG. 6A) or an anti-CXCL12 antibody (FIG.
6B), and inhibition of binding of .sup.125I-labeled CXCL12 to Ramos
cells by MDX-1338 (FIG. 6C). Ligand binding assays were conducted
by incubating 100 pM .sup.125I-CXCL12 with CEM cells in the
presence of increasing concentration of MDX-1338, anti-CXCL12, or
isotype control antibody. Unlabeled CXCL12 was added at 1000-fold
molar excess (100 nM) to establish non-specific binding (NSB).
.sup.125I-CXCL12 without antibody or unlabeled competitor was added
to establish total achievable binding (Total).
[0028] FIG. 7 shows inhibition of CXCL12 (SDF-1)-induced calcium
flux in CEM cells by anti-CXCR4 human antibodies F7 (BMS-936564),
F9 and D1. E2 does not significantly inhibit CXCL12-induced calcium
flux.
[0029] FIGS. 8A and 8B show inhibition of CXCL12-induced calcium
flux in CXCR4.sup.+ cells by anti-CXCR4 antibody MDX-1338
(BMS-936564) or an anti-CXCL12 antibody. Calcium flux assays were
conducted by incubating either Ramos cells (FIG. 8A) or CEM cells
(FIG. 8B) with Calcium 4 dye in the presence or absence of the test
antibody or an isotype control. Dye-loaded cells were incubated at
room temperature with 50 nM and 5 nM CXCL12 with the Ramos and CEM
cells, respectively. The area under the curve of fluorescence
between 20 to 200 seconds was quantitated and an EC.sub.50 was
calculated.
[0030] FIG. 9 shows inhibition of CXCL12-induced migration of CEM
cells by anti-CXCR4 human antibodies F7 (BMS-936564) and F9,
whereas antibodies D1 and E2 do not significantly inhibit
migration.
[0031] FIGS. 10A and 10B show inhibition of CXCL12-induced
migration of CXCR4.sup.+ cells by anti-CXCR4 antibody MDX-1338
(BMS-936564) or an anti-CXCL12 antibody. Migration assays with the
Ramos (FIG. 10A) and CEM (FIG. 10B) cells was carried out in the
presence of 1.25 nM and 0.05 nM CXCL12 respectively. The number of
labeled cells, which had migrated into the lower compartment, was
measured on a Fusion (PerkinElmer) plate reader. Each point
represents n=3.
[0032] FIGS. 11A and 11B: FIG. 11A shows the inhibition of Ramos
tumor cell proliferation in vitro by anti-CXCR4 human antibodies F7
(BMS-936564), F9 and E2, and FIG. 11B shows the inhibition of Ramos
cell proliferation by MDX-1338 (BMS-936564), compared to no
inhibition by anti-CXCL12. In FIG. 11B, the effects of various
peptide CXCR4 antagonists are also shown.
[0033] FIGS. 12A-12C show inhibition of Ramos tumor cell
proliferation in vivo in a subcutaneous tumor model by anti-CXCR4
human antibodies F7 (BMS-936564) and F9. FIG. 12A shows the mean
tumor volume growth curve; FIG. 12B shows the median tumor volume
growth curve; and FIG. 12C shows the median % body weight
change.
[0034] FIGS. 13A and 13B show percentage survival of mice treated
with the anti-CXCR4 human antibody F9 (FIG. 13A), or the anti-CXCR4
antibody, BMS-936564, and an anti-CXCL12 antibody (FIG. 13B) in a
Ramos systemic tumor cell model. BMS-936564 is highly efficacious
in this Ramos systemic model, whereas the anti-CXCL12 Ab shows no
efficacy.
[0035] FIGS. 14A and 14B show the results of an apoptosis assay
carried by incubating Ramos cells for 24 hours at 37.degree. C.
with 10 .mu.g/mL MDX-1338 (BMS-936564) or isotype control. Cells
were stained with Annexin V-FITC and propidium iodide (FIG. 14A).
The percent of cells positive for Annexin V only or both Annexin V
and PI double positive was determined (FIG. 14B).
[0036] FIGS. 15A and 15B show that induction of apoptosis by
MDX-1338 (BMS-936564) is CXCR4-specific. MDX-1338 or isotype
control were added to CXCR4-transfected cells (FIG. 15A) or R1610
parental cells (FIG. 15B) and stained with Annexin V-FITC and PI.
The percentages of cells that were positive for Annexin V only or
doubly positive for both Annexin V and PI double positive are
illustrated.
[0037] FIG. 16 shows in vivo tumor growth inhibition of a Ramos
cell lymphoma xenograft by a blocking CXCR4 antibody, MDX-1338
(BMS-936564), and a rituximab (chimeric anti-CD20 monoclonal
antibody) positive control, and the absence of tumor growth
inhibition by a blocking anti-CXCL12 antibody.
[0038] FIGS. 17A and 17B show in vivo tumor growth inhibition of a
HL60 cell (FIG. 17A) and a Nomo-1 (FIG. 17B) acute myeloid leukemia
xenograft by MDX-1338 (BMS-936564). Cytarabine expectedly did not
inhibit tumor growth of the cytarabine-resistant Nomo-1 tumor.
[0039] FIGS. 18A-18I show in vivo tumor growth inhibition of a
variety of CXCR4+ multiple myeloma cell xenografts by MDX-1338
(BMS-936564). FIG. 18A, tumor growth inhibition of MOLP8 cell
xenografts treated with MDX-1338 alone or in combination with
lenalidomide or bortezomib; FIG. 18B, tumor growth inhibition of
JJN-3R cell xenografts treated with MDX-1338 or lenalidomide or
bortezomib; FIG. 18C, tumor growth inhibition of parental JJN-3
cell xenografts treated with MDX-1338 alone or in combination with
bortezomib; FIG. 18D, tumor growth inhibition of parental JJN-3
cell xenografts treated with MDX-1338 alone or in combination with
lenalidomide; FIG. 18E, tumor growth inhibition of RPMI-8226 cell
xenografts by MDX-1338 alone or in combination with lenalidomide
(REVLIMID.RTM.); FIG. 18F, tumor growth inhibition of RPMI-8226
cell xenografts by MDX-1338 alone or in combination with bortezomib
(VELCADE.RTM.); FIG. 18G, tumor growth inhibition of MM.1S cell
xenografts by MDX-1338 alone or in combination with lenalidomide;
FIG. 18H, tumor growth inhibition of OMP-2 cell xenografts by
MDX-1338 alone or in combination with bortezomib; FIG. 18I, tumor
growth inhibition of OPM-2 cell xenografts by MDX-1338 alone or in
combination with lenalidomide.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present disclosure relates to isolated monoclonal
antibodies, particularly human monoclonal antibodies, which bind
specifically to native human CXCR4 expressed on a cell surface. In
certain embodiments, the antibodies of this disclosure are derived
from particular heavy and light chain germline sequences and/or
comprise particular structural features such as variable regions or
CDRs comprising particular amino acid sequences. This disclosure
also relates to methods of using the antibodies to modulate CXCR4
activity in, or otherwise treat, diseases or disorders associated
with expression of CXCR4 or involving the CXCR4/CXCL12 pathway,
such as cancers, particularly hematological malignancies, tumor
metastasis, HIV infection, inflammation and angiogenesis.
Terms
[0041] In order that the present disclosure may be more readily
understood, certain terms are first defined. As used in this
application, except as otherwise expressly provided herein, each of
the following terms shall have the meaning set forth below.
Additional definitions are set forth throughout the
application.
[0042] "Administering" refers to the physical introduction of a
composition comprising a therapeutic agent to a subject, using any
of the various methods and delivery systems known to those skilled
in the art. Preferred routes of administration for antibodies of
the invention include intravenous, intramuscular, subcutaneous,
intraperitoneal, spinal or other parenteral routes of
administration, for example by injection or infusion. The phrase
"parenteral administration" as used herein means modes of
administration other than enteral and topical administration,
usually by injection, and includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intralymphatic, intralesional, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion, as well as in vivo electroporation. Alternatively, an
antibody of the invention can be administered via a non-parenteral
route, such as a topical, epidermal or mucosal route of
administration, for example, intranasally, orally, vaginally,
rectally, sublingually or topically. Administering can also be
performed, for example, once, a plurality of times, and/or over one
or more extended periods.
[0043] An "antibody" (Ab) shall include, without limitation, a
glycoprotein immunoglobulin which binds specifically to an antigen
and comprises at least two heavy (H) chains and two light (L)
chains interconnected by disulfide bonds, or an antigen-binding
portion thereof. Each H chain comprises a heavy chain variable
region (abbreviated herein as V.sub.H) and a heavy chain constant
region. The heavy chain constant region comprises three domains,
C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain is comprised of a
light chain variable region (abbreviated herein as V.sub.L) and a
light chain constant region. The light chain constant region is
comprised of one domain, CL. The V.sub.H and V.sub.L 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 V.sub.H and V.sub.L is 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. The variable regions
of the heavy and light chains contain a binding domain that
interacts with an antigen. The constant regions of the antibodies
may mediate the binding of the immunoglobulin to host tissues or
factors, including various cells of the immune system (e.g.,
effector cells) and the first component (C1q) of the classical
complement system.
[0044] Antibodies typically bind specifically to their cognate
antigen with high affinity, reflected by a dissociation constant
(K.sub.D) of 10.sup.-5 to 10.sup.-11M.sup.-1 or less. Any K.sub.D
greater than about 10.sup.-4 M.sup.-1 is generally considered to
indicate nonspecific binding. As used herein, an antibody that
"binds specifically" to an antigen refers to an antibody that binds
to the antigen and substantially identical antigens with high
affinity, which means having a K.sub.D of 10.sup.-7 M or less,
preferably 10.sup.-8M or less, even more preferably
5.times.10.sup.-9M or less, and most preferably between 10.sup.-8M
and 10.sup.-10M or less, but does not bind with high affinity to
unrelated antigens. An antigen is "substantially identical" to a
given antigen if it exhibits a high degree of sequence identity to
the given antigen, for example, if it exhibits at least 80%, at
least 90%, preferably at least 95%, more preferably at least 97%,
or even more preferably at least 99 sequence identity to the
sequence of the given antigen. By way of example, an antibody that
binds specifically to human CXCR4 may also have cross-reactivity
with CXCR4 antigens from certain primate species but may not
cross-react with CXCR4 antigens from certain rodent species or with
an antigen other than CXCR4, e.g., a human PD-L1 antigen.
[0045] The immunoglobulin may derive from any of the commonly known
isotypes, including but not limited to IgA, secretory IgA, IgG and
IgM. IgG subclasses are also well known to those in the art and
include but are not limited to human IgG1, IgG2, IgG3 and IgG4.
"Isotype" refers to the antibody class (e.g., IgM or IgG1) that is
encoded by the heavy chain constant region genes. "Antibody"
includes, by way of example, both naturally occurring and
non-naturally occurring antibodies; monoclonal and polyclonal
antibodies; chimeric and humanized antibodies; human or nonhuman
antibodies; wholly synthetic antibodies; and single chain
antibodies. A nonhuman antibody may be humanized by recombinant
methods to reduce its immunogenicity in man. Where not expressly
stated, and unless the context indicates otherwise, the term
"antibody" also includes an antigen-binding fragment or an
antigen-binding portion of any of the aforementioned
immunoglobulins, and includes a monovalent and a divalent fragment
or portion, and a single chain antibody.
[0046] An "isolated antibody" refers to an antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated antibody that binds specifically
to CXCR4 is substantially free of antibodies that bind specifically
to antigens other than CXCR4). An isolated antibody that binds
specifically to CXCR4 may, however, have cross-reactivity to other
antigens, such as CXCR4 molecules from different species. Moreover,
an isolated antibody may be substantially free of other cellular
material and/or chemicals.
[0047] The phrases "an anti-antigen antibody", "an antibody
recognizing an antigen", and "an antibody specific for an antigen"
are used interchangeably herein with the term "an antibody which
binds specifically to an antigen."
[0048] The term "monoclonal antibody" ("mAb") refers to a
preparation of antibody molecules of single molecular composition,
i.e., antibody molecules whose primary sequences are essentially
identical, and which exhibits a single binding specificity and
affinity for a particular epitope. Monoclonal antibodies may be
produced by hybridoma, recombinant, transgenic or other techniques
known to those skilled in the art.
[0049] A "human" antibody (HuMAb) refers to an antibody having
variable regions in which both the framework and CDR regions are
derived from human germline immunoglobulin sequences. Furthermore,
if the antibody contains a constant region, the constant region
also is derived from human germline immunoglobulin sequences. The
human antibodies of the invention may include amino acid residues
not encoded by human germline immunoglobulin sequences (e.g.,
mutations introduced by random or site-specific mutagenesis in
vitro or by somatic mutation in vivo). However, the term "human
antibody", as used herein, is not intended to include antibodies in
which CDR sequences derived from the germline of another mammalian
species, such as a mouse, have been grafted onto human framework
sequences. The terms "human" antibodies and "fully human"
antibodies and are used synonymously.
[0050] A "humanized" antibody refers to an antibody in which some,
most or all of the amino acids outside the CDR domains of a
non-human antibody are replaced with corresponding amino acids
derived from human immunoglobulins. In one embodiment of a
humanized form of an antibody, some, most or all of the amino acids
outside the CDR domains have been replaced with amino acids from
human immunoglobulins, whereas some, most or all amino acids within
one or more CDR regions are unchanged. Small additions, deletions,
insertions, substitutions or modifications of amino acids are
permissible as long as they do not abrogate the ability of the
antibody to bind to a particular antigen. A "humanized" antibody
retains an antigenic specificity similar to that of the original
antibody.
[0051] A "chimeric antibody" refers to an antibody in which the
variable regions are derived from one species and the constant
regions are derived from another species, such as an antibody in
which the variable regions are derived from a mouse antibody and
the constant regions are derived from a human antibody.
[0052] An "antigen-binding portion" of an antibody (also called an
"antigen-binding fragment") refers to one or more fragments of an
antibody that retain the ability to bind specifically to the
antigen bound by the whole antibody.
[0053] A "cancer" refers a broad group of various diseases
characterized by the uncontrolled growth of abnormal cells in the
body. Unregulated cell division and growth divide and grow results
in the formation of malignant tumors that invade neighboring
tissues and may also metastasize to distant parts of the body
through the lymphatic system or bloodstream.
[0054] The term "CXCR4" ("C-X-C chemokine receptor 4") includes
variants, isoforms, homologs, orthologs and paralogs. For example,
antibodies specific for CXCR4 may, in certain cases, cross-react
with CXCR4 from species other than human. In other embodiments, the
antibodies specific for human CXCR4 may be completely specific for
human CXCR4 and may not exhibit species or other types of
cross-reactivity. The term "human CXCR4" refers to human sequence
CXCR4, such as the complete amino acid sequence of human CXCR4
having GENBANK.RTM. accession number P61073 (SEQ ID NO: 51). CXCR4
is also known in the art as, for example, LESTR, Fusin or CD184.
The human CXCR4 sequence may differ from human CXCR4 of SEQ ID NO:
51 by having, for example, conserved mutations or mutations in
non-conserved regions, and the CXCR4 has substantially the same
biological function as the human CXCR4 of SEQ ID NO: 51. For
example, a biological function of human CXCR4 is having an epitope
in the extracellular domain of CXCR4 that is specifically bound by
an antibody of the instant disclosure or the biological function of
human CXCR4 is chemokine binding or involvement in the metastatic
process.
[0055] A particular human CXCR4 sequence will generally be at least
90% identical in amino acids sequence to human CXCR4 of SEQ ID NO:
51 and contains amino acid residues that identify the amino acid
sequence as being human when compared to CXCR4 amino acid sequences
of other species (e.g., murine). In certain cases, a human CXCR4
may be at least 95%, or even at least 96%, 97%, 98%, or 99%
identical in amino acid sequence to CXCR4 of SEQ ID NO: 51. In
certain embodiments, a human CXCR4 sequence will display no more
than 10 amino acid differences from the CXCR4 of SEQ ID NO: 51. In
certain embodiments, the human CXCR4 may display no more than 5, or
even no more than 4, 3, 2, or 1 amino acid difference from the
CXCR4 of SEQ ID NO: 51. Percent identity can be determined as
described herein.
[0056] A "CXCR4-expressing cancer" or "CXCR4.sup.+ cancer" is a
cancer wherein the malignant cells that characterize this cancer
express CXCR4 on the cell surface, preferably expressing a high
level of CXCR4.
[0057] The term "hematological malignancy" herein includes a
lymphoma, leukemia, myeloma or a lymphoid malignancy, as well as a
cancer of the spleen and the lymph nodes. Exemplary lymphomas that
are amenable to treatment with the disclosed anti-CXCR4 antibodies
of this invention include both B cell lymphomas and T cell
lymphomas. B-cell lymphomas include both Hodgkin's lymphomas and
most non-Hodgkins lymphomas. Non-limiting examples of B cell
lymphomas include diffuse large B-cell lymphoma (DLBCL), follicular
lymphoma (FL), mucosa-associated lymphatic tissue lymphoma (MALT),
small cell lymphocytic lymphoma (overlaps with chronic lymphocytic
leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma,
mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia,
nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone
lymphoma (SMZL), intravascular large B-cell lymphoma, primary
effusion lymphoma, lymphomatoid granulomatosis. Non-limiting
examples of T cell lymphomas include extranodal T cell lymphoma,
cutaneous T cell lymphomas, anaplastic large cell lymphoma, and
angioimmunoblastic T cell lymphoma. Hematological malignancies also
include leukemia, such as, but not limited to, secondary leukemia,
chronic lymphocytic leukemia (CLL; also called chronic lymphoid
leukemia), acute myelogenous leukemia (AML; also called acute
lymphoid leukemia), chronic myelogenous leukemia (CIVIL), B-cell
prolymphocytic leukemia (B-PLL), acute lymphoblastic leukemia (ALL)
and myelodysplasia (MDS). Hematological malignancies further
include myelomas, such as, but not limited to, multiple myeloma
(MM) and smoldering multiple myeloma (SMM). Other hematological
and/or B cell- or T-cell-associated cancers are encompassed by the
term hematological malignancy. For example, hematological
malignancies also include cancers of additional hematopoietic
cells, including dendritic cells, platelets, erythrocytes, natural
killer cells, and polymorphonuclear leukocytes, e.g., basophils,
eosinophils, neutrophils and monocytes. It should be clear to those
of skill in the art that these pre-malignancies and malignancies
will often have different names due to changing systems of
classification, and that patients having lymphomas classified under
different names may also benefit from the therapeutic regimens of
the present invention.
[0058] The term "SDF-1" refers to stromal cell-derived factor 1,
which is a ligand for CXCR4. The term "SDF-1" encompasses different
isoforms of SDF-1, such as SDF-1.alpha. and SDF-1.beta.. The amino
acid sequence of human SDF-1.alpha. has GENBANK.RTM. accession
number NP_954637. The amino acid sequence of human SDF-1.beta. has
GENBANK.RTM. accession number NP_000600. Human SDF-1 is also
described in U.S. Pat. No. 5,756,084. SDF-1 is also known as
CXCL12. The amino acid sequence of human SDF-1 can differ from the
SDF-1 of NP_954637 or NP_000600, as described herein for CXCR4.
[0059] A "signal transduction pathway" refers to the biochemical
relationship between a variety of signal transduction molecules
that play a role in the transmission of a signal from one portion
of a cell to another portion of a cell. As used herein, the phrase
"cell surface receptor" includes, for example, molecules and
complexes of molecules capable of receiving a signal and the
transmission of such a signal across the plasma membrane of a cell.
An example of a cell surface receptor of the present disclosure is
the CXCR4 receptor.
[0060] A "subject" includes any human or nonhuman animal. The term
"nonhuman animal" includes, but is not limited to, vertebrates such
as nonhuman primates, sheep, dogs, cats, rabbits, ferrets, rodents
such as mice, rats and guinea pigs, avian species such as chickens,
amphibians, and reptiles. In preferred embodiments, the subject is
a mammal such as a nonhuman primate, sheep, dog, cat, rabbit,
ferret or rodent. In more preferred embodiments, the subject is a
human. The terms, "subject", "patient" and "individual" are used
interchangeably herein.
[0061] A "therapeutically effective amount" or "therapeutically
effective dosage" of a drug or therapeutic agent, such as an
antibody of the invention, is any amount of the drug that, when
used alone or in combination with another therapeutic agent,
promotes disease regression evidenced by a decrease in severity of
disease symptoms, an increase in frequency and duration of disease
symptom-free periods, or a prevention of impairment or disability
due to the disease affliction. A therapeutically effective amount
or dosage of a drug includes a "prophylactically effective amount"
or a "prophylactically effective dosage", which is any amount of
the drug that, when administered alone or in combination with
another therapeutic agent to a subject at risk of developing a
disease or of suffering a recurrence of disease, inhibits the
development or recurrence of the disease. The ability of a
therapeutic agent to promote disease regression can be evaluated
using a variety of methods known to the skilled practitioner, such
as in human subjects during clinical trials, in animal model
systems predictive of efficacy in humans, or by assaying the
activity of the agent in in vitro assays.
[0062] By way of example, an anti-cancer agent promotes cancer
regression in a subject. In preferred embodiments, a
therapeutically effective amount of the drug promotes cancer
regression to the point of eliminating the cancer. "Promoting
cancer regression" means that administering an effective amount of
the drug, alone or in combination with an anti-neoplastic agent,
results in a reduction in tumor growth or size, necrosis of the
tumor, a decrease in severity of at least one disease symptom, an
increase in frequency and duration of disease symptom-free periods,
a prevention of impairment or disability due to the disease
affliction, or otherwise amelioration of disease symptoms in the
patient. In addition, the terms "effective" and "effectiveness"
with regard to a treatment includes both pharmacological
effectiveness and physiological safety. Pharmacological
effectiveness refers to the ability of the drug to promote cancer
regression in the patient. Physiological safety refers to the level
of toxicity, or other adverse physiological effects at the
cellular, organ and/or organism level (adverse effects) resulting
from administration of the drug.
[0063] By way of example for the treatment of tumors, a
therapeutically effective amount or dosage of the drug preferably
inhibits cell growth or tumor growth by at least about 20%, more
preferably by at least about 40%, even more preferably by at least
about 60%, and still more preferably by at least about 80% relative
to untreated subjects. In the most preferred embodiments, a
therapeutically effective amount or dosage of the drug completely
inhibits cell growth or tumor growth, i.e., preferably inhibits
cell growth or tumor growth by 100%. The ability of a compound to
inhibit tumor growth can be evaluated in an animal model system
predictive of efficacy in human tumors. Alternatively, this
property of a composition can be evaluated by examining the ability
of the compound to inhibit cell growth, such inhibition can be
measured in vitro by assays known to the skilled practitioner. In
other preferred embodiments of the invention, tumor regression may
be observed and continue for a period of at least about 20 days,
more preferably at least about 40 days, or even more preferably at
least about 60 days.
[0064] "Treatment" or "therapy" of a subject refers to any type of
intervention or process performed on, or administering an active
agent to, the subject with the objective of reversing, alleviating,
ameliorating, inhibiting, slowing down or prevent the onset,
progression, development, severity or recurrence of a symptom,
complication, condition or biochemical indicia associated with a
disease.
[0065] Various aspects of this disclosure are described in further
detail in the following subsections.
Anti-CXCR4 Antibodies
[0066] Human monoclonal anti-CXCR4 antibodies of this disclosure
can be generated using transgenic or transchromosomic mice carrying
parts of the human immune system rather than the mouse system.
These transgenic and transchromosomic mice include mice referred to
herein as the HUMAB MOUSE.RTM. (Lonberg et al., 1994) and KM
MOUSE.RTM. (WO 02/43478), respectively. The production of exemplary
anti-CXCR4 antibodies of this invention is described in detail in
WO 2008/060367. The antibodies of this disclosure are characterized
by particular functional features or properties. For example, the
antibodies bind to native human CXCR4 expressed on a cell surface.
Preferably, an antibody of this disclosure binds to CXCR4 with high
affinity, for example with a K.sub.D of 1.times.10.sup.-7 M or
less. The anti-CXCR4 antibodies of this disclosure preferably
exhibit one or more of the following characteristics: [0067] (a)
binding to native human CXCR4 expressed on a cell surface; [0068]
(b) inhibiting binding of SDF-1 to CXCR4; [0069] (c) inhibiting
SDF-1-induced calcium flux in cells expressing CXCR4; [0070] (d)
inhibiting SDF-1-induced migration of cells expressing CXCR4;
[0071] (e) inhibiting capillary tube formation by human umbilical
vein endothelial cells; [0072] (f) binding to human CXCR4 with a KD
of 1.times.10.sup.-7 M or less; [0073] (g) inducing apoptosis in
cells expressing CXCR4; [0074] (h) inhibiting proliferation of
CXCR4.sup.+ tumor cells in vitro; [0075] (i) inhibiting CXCR4.sup.+
tumor cell proliferation and/or inducing CXCR4.sup.+ tumor cell
apoptosis in vivo;
[0076] (j) inhibiting metastases of CXCR4.sup.+ tumor cells; and/or
[0077] (k) increasing survival time of a CXCR4.sup.+ tumor-bearing
subject.
[0078] Preferably, an antibody of this disclosure binds to human
CXCR4 with a K.sub.D of 5.times.10.sup.-8M or less, binds to human
CXCR4 with a K.sub.D of 2.times.10.sup.-8M or less, binds to human
CXCR4 with a K.sub.D of 5.times.10.sup.-9M or less, binds to human
CXCR4 with a K.sub.D of 4.times.10.sup.-9M or less, binds to human
CXCR4 with a K.sub.D of 3.times.10.sup.-9M or less, or binds to
human CXCR4 with a K.sub.D of 2.times.10.sup.-9M or less.
[0079] Preferably, an antibody of the inhibits binding of SDF-1 to
human CXCR4 with an EC.sub.50 for inhibition of 50 nM or less, more
preferably 30 nM or less, or 15 nM or less, or 10 nM or less, or 5
nM or less, or 3 nM or less (e.g., an EC.sub.50 for inhibition of
28.60 nM or less, or 12.51 nM or less, or 2.256 nM or less)
[0080] Preferably, an antibody of this disclosure inhibits
SDF-1-induced calcium flux in cells expressing human CXCR4 with an
EC.sub.50 for inhibition of 3 nM or less, more preferably 2 nM or
less, or 1 nM or less, or 0.9 nM or less, or 0.8 nM or less, or 0.7
nM or less, or 0.6 nM or less, or 0.5 nM or less, or 0.4 nM or less
(e.g., 0.9046 nM or less, 0.5684 or less, or 0.3219 nM or
less).
[0081] Preferably, an antibody of this disclosure inhibits
SDF-1-induced migration of cells expressing human CXCR4 with an
EC.sub.50 for inhibition of 50 nM or less, more preferably 30 nM or
less, or 20 nM or less, or 15 nM or less (e.g., 18.99 nM or less,
or 12.44 or less).
[0082] Standard assays to evaluate the binding ability of the
antibodies toward native human CXCR4 expressed on a cell surface
are known in the art, including for example, flow cytometry
analysis using a cell line that naturally expresses native CXCR4 or
that has been transfected to express native CXCR4. Suitable assays
are described in detail in the Examples. A preferred cell line that
expresses native CXCR4 is the CEM T cell line. Suitable assays for
evaluating inhibition of binding of SDF-1, inhibition of SDF-1
induced calcium flux, inhibition of SDF-1 induced cell migration,
inhibition of capillary tube formation by HuVECs, induction of
apoptosis in cells expressing CXCR4 in vitro and/or in vivo,
inhibition of growth of CXCR4.sup.+ tumor cells in vitro and/or in
vivo, and/or inhibition of metastases of CXCR4.sup.+ tumor cells
are also described in detail in the Examples. Binding affinity of
the antibodies also can be determined by standard methods, such as
by Scatchard analysis.
[0083] Anti-CXCR4 antibodies of the invention also include
antigen-binding portions of the above antibodies. It has been amply
demonstrated that the antigen-binding function of an antibody can
be performed by fragments of a full-length antibody. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the V.sub.H and
C.sub.H1 domains; and (iv) a Fv fragment consisting of the V.sub.L
and V.sub.H domains of a single arm of an antibody.
[0084] These fragments, obtained initially through proteolysis with
enzymes such as papain and pepsin, have been subsequently
engineered into monovalent and multivalent antigen-binding
fragments. For example, although the two domains of the Fv
fragment, V.sub.L and V.sub.H, are coded for by separate genes,
they can be joined, using recombinant methods, by a synthetic
linker peptide that enables them to be made as a single protein
chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules known as single chain variable fragments
(scFv). Divalent or bivalent scFvs (di-scFvs or bi-scFvs) can be
engineered by linking two scFvs in within a single peptide chain
known as a tandem scFv which contains two V.sub.H and two V.sub.L
regions. ScFv dimers and higher multimers can also be created using
linker peptides of fewer than 10 amino acids that are too short for
the two variable regions to fold together, which forces the scFvs
to dimerize and produce diabodies or form other multimers.
Diabodies have been shown to bind to their cognate antigen with
much higher affinity than the corresponding scFvs, having
dissociation constants up to 40-fold lower than the K.sub.D values
for the scFvs. Very short linkers (<3 amino acids) lead to the
formation of trivalent triabodies or tetravalent tetrabodies that
exhibit even higher affinities for to their antigens than
diabodies. Other variants include minibodies, which are
scFv-C.sub.H3 dimers, and larger scFv-Fc fragments
(scFv-C.sub.H2-C.sub.H3 dimers), and even an isolated CDR may
exhibit antigen-binding function. These antibody fragments are
engineered using conventional recombinant techniques known to those
of skill in the art, and the fragments are screened for utility in
the same manner as are intact antibodies. All of the above
proteolytic and engineered fragments of antibodies and related
variants (see Hollinger et al., 2005; Olafsen et al., 2010, for
further details) are intended to be encompassed within the term
"antigen-binding portion" of an antibody.
Monoclonal Antibodies F7, F9, D1 and E2
[0085] Preferred antibodies of this disclosure are the human
monoclonal antibodies F7 (BMS-936564), F9, D1 and E2, isolated and
structurally characterized as described in Examples 1 and 2. The
V.sub.H amino acid sequences of F7, F9, D1 and E2 are shown in SEQ
ID NOs. 25, 26, 27 and 28, respectively. The V.sub.L amino acid
sequences of F7, F9, D1 and E2 are shown in SEQ ID NOs. 29, 30, 31
and 32, respectively. Additionally, alternative forms of F7, F9, D1
and E2, in which certain framework residues were substituted with a
germline residue, were created and are referred to herein as F7GL,
F9GL, D1GL and E2GL. The V.sub.H amino acid sequences of F7GL,
F9GL, D1GL and E2GL are shown in SEQ ID NOs. 41, 42, 43 and 44,
respectively. The V.sub.L amino acid sequences of F7GL, F9GL, D1GL
and E2GL are shown in SEQ ID NOs. 45, 46, 47 and 48, respectively.
Other anti-CXCR4 antibodies of this disclosure include antibodies
result from "mixing and matching" different V.sub.H and V.sub.L
regions, or different CDRs, to create antibodies that bind
specifically to CXCR4 as described in WO 2008/060367.
[0086] Accordingly, in one aspect, this disclosure provides
antibodies that comprise the heavy chain and light chain CDR1's,
CDR2's and CDR3's of F7, F9, D1 or E2, or combinations thereof. The
amino acid sequences of the V.sub.H CDR1's of F7, F9, D1 and E2 are
shown in SEQ ID NOs. 1-4, respectively. The amino acid sequences of
the V.sub.H CDR2's of F7, F9, D1 and E2 are shown in SEQ ID NOs.
5-8, respectively. The amino acid sequences of the V.sub.H CDR3's
of F7, F9, D1 and E2 are shown in SEQ ID NOs. 9-12, respectively.
The amino acid sequences of the V.sub.k CDR1's of F7, F9, D1 and E2
are shown in SEQ ID NOs. 13-16, respectively. The amino acid
sequences of the V.sub.k CDR2's of F7, F9, D1 and E2 are shown in
SEQ ID NOs. 17-20, respectively. The amino acid sequences of the
V.sub.k CDR3's of F7, F9, D1 and E2 are shown in SEQ ID NOs. 21-24,
respectively. The CDR regions identified above were delineated
using the Kabat system (Kabat et al., 1991).
[0087] In one aspect, this disclosure provides a monoclonal
antibody or antigen-binding portion thereof which binds
specifically to CXCR4, preferably human CXCR4, and comprises a
combination of V.sub.H and V.sub.L regions, each comprising three
complementarity-determining regions (CDRs). In preferred
embodiments, the monoclonal antibody or antigen-binding portion
thereof comprises:
[0088] (a) the CDR1, CDR2 and CDR3 domains in a heavy chain
variable region having the sequence set forth in SEQ ID NO: 25 or
41, and the CDR1, CDR2 and CDR3 domains in a light chain variable
region having the sequence set forth in SEQ ID NO: 29 or 45;
[0089] (b) the CDR1, CDR2 and CDR3 domains in a heavy chain
variable region having the sequence set forth in SEQ ID NO: 26 or
42, and the CDR1, CDR2 and CDR3 domains in a light chain variable
region having the sequence set forth in SEQ ID NO: 30 or 46;
[0090] (c) the CDR1, CDR2 and CDR3 domains in a heavy chain
variable region having the sequence set forth in SEQ ID NO: 27 or
43, and the CDR1, CDR2 and CDR3 domains in a light chain variable
region having the sequence set forth in SEQ ID NO: 31 or 47; or
[0091] (d) the CDR1, CDR2 and CDR3 domains in a heavy chain
variable region having the sequence set forth in SEQ ID NO: 28 or
44, and the CDR1, CDR2 and CDR3 domains in a light chain variable
region having the sequence set forth in SEQ ID NO: 32 or 48.
[0092] In other preferred embodiments, the monoclonal antibody or
antigen-binding portion thereof of the invention comprises:
[0093] (a) a heavy chain variable region CDR1 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 1 or conservative modifications thereof; a heavy chain
variable region CDR2 comprising consecutively linked amino acids
having the sequence set forth in SEQ ID NO: 5 or conservative
modifications thereof; a heavy chain variable region CDR3
comprising consecutively linked amino acids having the sequence set
forth in SEQ ID NO: 9 or conservative modifications thereof; a
light chain variable region CDR1 comprising consecutively linked
amino acids having the sequence set forth in SEQ ID NO: 13 or
conservative modifications thereof; a light chain variable region
CDR2 comprising consecutively linked amino acids having the
sequence set forth in SEQ ID NO: 17 or conservative modifications
thereof; and a light chain variable region CDR3 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 21;
[0094] (b) a heavy chain variable region CDR1 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 2 or conservative modifications thereof; a heavy chain
variable region CDR2 comprising consecutively linked amino acids
having the sequence set forth in SEQ ID NO: 6 or conservative
modifications thereof; a heavy chain variable region CDR3
comprising consecutively linked amino acids having the sequence set
forth in SEQ ID NO: 10 or conservative modifications thereof; a
light chain variable region CDR1 comprising consecutively linked
amino acids having the sequence set forth in SEQ ID NO: 14 or
conservative modifications thereof; a light chain variable region
CDR2 comprising consecutively linked amino acids having the
sequence set forth in SEQ ID NO: 18 or conservative modifications
thereof; and a light chain variable region CDR3 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 22;
[0095] (c) a heavy chain variable region CDR1 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 3 or conservative modifications thereof; a heavy chain
variable region CDR2 comprising consecutively linked amino acids
having the sequence set forth in SEQ ID NO: 7 or conservative
modifications thereof; a heavy chain variable region CDR3
comprising consecutively linked amino acids having the sequence set
forth in SEQ ID NO: 11 or conservative modifications thereof; a
light chain variable region CDR1 comprising consecutively linked
amino acids having the sequence set forth in SEQ ID NO: 15 or
conservative modifications thereof; a light chain variable region
CDR2 comprising consecutively linked amino acids having the
sequence set forth in SEQ ID NO: 19 or conservative modifications
thereof; and a light chain variable region CDR3 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 23; or
[0096] (d) a heavy chain variable region CDR1 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 4 or conservative modifications thereof; a heavy chain
variable region CDR2 comprising consecutively linked amino acids
having the sequence set forth in SEQ ID NO: 8 or conservative
modifications thereof; a heavy chain variable region CDR3
comprising consecutively linked amino acids having the sequence set
forth in SEQ ID NO: 12 or conservative modifications thereof; a
light chain variable region CDR1 comprising consecutively linked
amino acids having the sequence set forth in SEQ ID NO: 16 or
conservative modifications thereof; a light chain variable region
CDR2 comprising consecutively linked amino acids having the
sequence set forth in SEQ ID NO: 20 or conservative modifications
thereof; and a light chain variable region CDR3 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 24.
[0097] In further embodiments, the monoclonal antibody or
antigen-binding portion thereof of the invention comprises:
[0098] (a) a heavy chain variable region comprising consecutively
linked amino acids having the sequence set forth in SEQ ID NO: 25
or 41 or conservative modifications thereof, and a light chain
variable region comprising consecutively linked amino acids having
the sequence set forth in SEQ ID NO: 29 or 45 or conservative
modifications thereof;
[0099] (b) a heavy chain variable region comprising consecutively
linked amino acids having the sequence set forth in SEQ ID NO: 26
or 42 or conservative modifications thereof, and a light chain
variable region comprising consecutively linked amino acids having
the sequence set forth in SEQ ID NO: 30 or 46 or conservative
modifications thereof;
[0100] (c) a heavy chain variable region comprising consecutively
linked amino acids having the sequence set forth in SEQ ID NO: 27
or 43 or conservative modifications thereof, and a light chain
variable region comprising consecutively linked amino acids having
the sequence set forth in SEQ ID NO: 31 or 47 or conservative
modifications thereof; or
[0101] (d) a heavy chain variable region comprising consecutively
linked amino acids having the sequence set forth in SEQ ID NO: 28
or 44 or conservative modifications thereof, and a light chain
variable region comprising consecutively linked amino acids having
the sequence set forth in SEQ ID NO: 32 or 48 or conservative
modifications thereof.
[0102] In a preferred embodiment, the anti-CXCR4 antibody or
antigen-binding portion thereof comprises:
[0103] (a) a heavy chain variable region CDR1 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 1;
[0104] (b) a heavy chain variable region CDR2 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 5;
[0105] (c) a heavy chain variable region CDR3 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 9;
[0106] (d) a light chain variable region CDR1 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 13;
[0107] (e) a light chain variable region CDR2 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 17; and a light chain variable region CDR3 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 21.
[0108] In another preferred embodiment, the anti-CXCR4 antibody or
antigen-binding portion thereof comprises:
[0109] (a) a heavy chain variable region CDR1 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 2;
[0110] (b) a heavy chain variable region CDR2 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 6;
[0111] (c) a heavy chain variable region CDR3 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 10;
[0112] (d) a light chain variable region CDR1 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 14;
[0113] (e) a light chain variable region CDR2 comprising
consecutively linked amino acids having the sequence set forth in
SEQ ID NO: 18; and [0114] (f) a light chain variable region CDR3
comprising consecutively linked amino acids having the sequence set
forth in SEQ ID NO: 22. Antibodies that Bind to the Same Epitope as
Anti-CXCR4 Antibodies
[0115] In another embodiment, this disclosure provides antibodies
or antigen-binding portions thereof that bind to the same epitope
region (i.e., the same or an overlapping epitope) on human CXCR4 as
any of the anti-CXCR4 monoclonal antibodies of this disclosure
(i.e., antibodies that have the ability to cross-compete for
binding to CXCR4 with any of the monoclonal antibodies of this
disclosure). In preferred embodiments, the reference antibody for
cross-competition studies can be the monoclonal antibody F7
(BMS-936564) (having V.sub.H and V.sub.L sequences as shown in SEQ
ID NOs: 25 and 29, respectively), or the monoclonal antibody F9
(having V.sub.H and V.sub.L sequences as shown in SEQ ID NOs: 26
and 30, respectively) or the monoclonal antibody D1 (having V.sub.H
and V.sub.L sequences as shown in SEQ ID NOs: 27 and 31,
respectively) or the monoclonal antibody E2 (having V.sub.H and
V.sub.L sequences as shown in SEQ ID NOs: 28 and 32, respectively).
Accordingly, this disclosure provides a human monoclonal antibody,
or an antigen-binding portion thereof, which cross-competes for
binding to human CXCR4 with a reference antibody or reference
antigen-binding portion thereof, wherein the reference antibody or
portion thereof comprises:
[0116] (a) a heavy chain variable region comprising consecutively
linked amino acids having the sequence set forth in SEQ ID NO: 25
and a light chain variable region comprising consecutively linked
amino acids having the sequence set forth in SEQ ID NO: 29;
[0117] (b) a heavy chain variable region comprising consecutively
linked amino acids having the sequence set forth in SEQ ID NO: 26
and a light chain variable region comprising consecutively linked
amino acids having the sequence set forth in SEQ ID NO: 30;
[0118] (c) a heavy chain variable region comprising consecutively
linked amino acids having the sequence set forth in SEQ ID NO: 27
and a light chain variable region comprising consecutively linked
amino acids having the sequence set forth in SEQ ID NO: 31; or
[0119] (d) a heavy chain variable region comprising consecutively
linked amino acids having the sequence set forth in SEQ ID NO: 28
and a light chain variable region comprising consecutively linked
amino acids having the sequence set forth in SEQ ID NO: 32.
[0120] In a preferred aspect, the cross-competing anti-CXCR4
monoclonal antibody of the invention comprises a V.sub.H region
comprising consecutively linked amino acids having a sequence
derived from a human V.sub.H 3-48 germline sequence as set forth in
SEQ ID NO: 49 and/or a V.sub.L region comprising consecutively
linked amino acids having a sequence derived from a human V.sub.K
L15 germline sequence as set forth in SEQ ID NO: 50.
[0121] The cross-competing antibodies can be identified based on
their ability to cross-compete with F7, F9, D1, E2 or any other
reference anti-CXCR4 antibody of the invention in a standard CXCR4
binding assay, for example, flow cytometry with CEM cells, wherein
the reference antibody is labeled with FITC and the ability of a
test antibody to inhibit the binding of the FITC-labeled reference
antibody to CEM cells is evaluated.
Pharmaceutical Compositions
[0122] In another aspect, the present disclosure provides a
composition, e.g., a pharmaceutical composition, containing one or
a combination of monoclonal antibodies, or antigen-binding
portion(s) thereof, of the present disclosure, formulated together
with a pharmaceutically acceptable carrier. As used herein, a
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Preferably, the carrier is suitable
for intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal administration (e.g., by injection or infusion). A
pharmaceutical composition of the invention may include one or more
pharmaceutically acceptable salts, anti-oxidant, aqueous and
nonaqueous carriers, and/or adjuvants such as preservatives,
wetting agents, emulsifying agents and dispersing agents.
[0123] Dosage regimens are adjusted to provide the optimum desired
response, e.g., a therapeutic response or minimal adverse
effects.
[0124] For administration of a human anti-CXCR4 antibody, the
dosage ranges from about 0.0001 to 100 mg/kg, preferably from about
0.01 to about 20 mg/kg, and more preferably 0.1 to 10 mg/kg, of the
subject's body weight. For example, dosages can be 0.1, 0.3, 1, 3,
5 or 10 mg/kg body weight, and more preferably, 0.3, 1, 3, or 10
mg/kg body weight. The dosing schedule is typically designed to
achieve exposures that result in sustained receptor occupancy based
on typical pharmacokinetic properties of an antibody. An exemplary
treatment regime entails administration once per week, once every
two weeks, once every three weeks, once every four weeks, once a
month, once every 3 months or once every three to 6 months.
Considering that an IgG4 antibody typically has a half-life of 2-3
weeks, a preferred dosage regimen for an anti-CXCR4 antibody of the
disclosure comprises 0.3-20 mg/kg body weight, preferably 1-10
mg/kg body weight, via intravenous administration, with the
antibody being given every 7 or 14 days in up to 6-week, 8-week or
12-week cycles until complete response or confirmed progressive
disease.
[0125] The dosage and scheduling may change during a course of
treatment. For example, dosage regimens for an anti-CXCR4 antibody
of this disclosure include 1, 3 or 10 mg/kg body weight via
intravenous (IV) administration, with the antibody being given
using one of the following dosing schedules: (i) every 7 days in up
to 6-week cycles; (ii) every two weeks for up to six dosages, then
every three months; (iii) every three weeks; (iv) 1-10 mg/kg body
weight once followed by 1 mg/kg body weight every 2-3 weeks.
[0126] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months or yearly. Intervals can also
be irregular as indicated by measuring blood levels of antibody to
the target antigen in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of about 1-1000
.mu.g/ml and in some methods about 25-300 .mu.g/ml.
[0127] Alternatively, antibody can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0128] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present disclosure may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present disclosure
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts. A composition of the present invention can be
administered via one or more routes of administration using one or
more of a variety of methods well known in the art. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
[0129] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Robinson (1978).
[0130] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of this disclosure can be administered with
a needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, or 4,941,880. The
subject matter of these patents is incorporated herein by
reference. Many other such implants, delivery systems, and modules
are known to those skilled in the art.
Uses and Methods of the Invention
[0131] The antibodies, antibody compositions and methods of the
present disclosure have numerous in vitro and in vivo diagnostic
and therapeutic utilities involving the diagnosis and treatment of
CXCR4-associated disorders including, for example, methods for
treating a subject afflicted with a CXCR4-expressing cancer
comprising administering to the subject a therapeutically effective
amount of an antibody or a fragment thereof that specifically binds
to CXCR4 expressed on a cell surface. Preferred subjects include
human patients having disorders such as hematological malignancies
that are associated with, mediated or modulated by, CXCR4 activity
or involve the CXCR4/CXCL12 pathway. In certain embodiments of
these methods for treating a cancer patient, the anti-CXCR4
antibody or fragment thereof is administered as monotherapy,
whereas in other embodiments, it is administered in combination
with another agent, such as an anti-neoplastic chemotherapeutic
agent. When antibodies to CXCR4 are administered in combination
with another agent, the two can be administered in either order or
simultaneously.
[0132] CXCR4 is known to be expressed on a wide variety of tumor
cells types and also is known to be involved in tumor metastasis.
Moreover, as a coreceptor for HIV entry into T cells, CXCR4 is
known to be involved in HIV infection. Additionally, the
CXCR4/CXCL12 pathway has been shown to be involved in inflammatory
conditions. Still further, the CXCR4/CXCL12 pathway has been shown
to be involved in angiogenesis or neovascularization. Accordingly,
the anti-CXCR4 antibodies (and immunoconjugates and bispecific
molecules) of this disclosure can be used in a variety of clinical
situations, including the following:
[0133] A. Cancer
[0134] Over-expression of CXCR4 has also been demonstrated in about
75% of cancers, and in certain situations an inverse correlation
has been established between CXCR4 expression and patient prognosis
or survival. Non-limiting examples of cancer types associated with
CXCR4 expression or the CXCR4/CXCL12 pathway include solid tumors
such as breast (Muller et al., 2001), ovarian (Scotton et al.,
2001), prostate (Taichman et al., 2002), non-small cell lung (Spano
et al., 2004), pancreatic (Koshiba et al., 2000), colorectal
(Zeelenberg et al., 2003), kidney (Schrader et al., 2002), and
thyroid cancer (Hwang et al., 2003), nasopharyngeal carcinoma (Wang
et al., 2005), melanoma (Scala et al., 2005), renal cell carcinoma
(Staller et al., 2003), neuroblastoma (Geminder et al., 2001),
glioblastoma (Rempel et al., 2000), rhabdomyosarcoma (Libura et
al., 2002), and osteosarcoma (Laverdiere et al., 2005), as well as
hematological malignancies such as acute lymphoblastic leukemia
(Crazzolara et al., 2001), acute myeloid leukemia (Mohle et al.,
1998; Rombouts et al., 2004), multiple myeloma (Alsayed et al.,
2007; Azab et al., 2009), chronic lymphoid leukemia (Mohle et al.,
1999; Burger et al., 1999), chronic myeloid leukemia (Jin et al.,
2008), and non-Hodgkin's lymphoma (Bertolini et al., 2002; Weng et
al., 2003).
[0135] Additionally, this pathway is implicated in stimulating the
metastatic process in multiple neoplasms (Murphy, 2001). In
clinical studies, CXCR4 has been associated with increased
propensity for metastasis and decreased survival and has been
identified as a prognostic indicator for acute myeloid leukemia,
breast, colorectal, non-small-cell lung, ovarian and pancreatic
carcinoma in which greater expression of CXCR4 correlates with
disease severity (Spoo et al., 2007; Hiller et al., 2011; Ottaiano
et al., 2006; Spano et al., 2004; Jiang et al.; 2006; Marechal et
al., 2009).
[0136] Bone marrow stromal cells (BMSCs) secrete CXCL12 and the
interaction with CXCR4 is essential for homing and maintaining
hematopoietic stem cells within the BM microenvironment (Mohle et
al., 1998). Leukemic cells express high levels of CXCR4, and the
pathway plays a critical role in leukemic cell migration into the
BM which in turn, supports their growth and survival. CXCR4 is
essential for metastatic spread to organs such as BM where CXCL12
is expressed. Collectively, CXCR4 plays an important role in both
homing and retention of hematopoietic stem cells in the BM and an
antagonist of CXCR4 mobilizes stem cells into the bloodstream, as
demonstrated with the small-molecule CXCR4 antagonist, AMD3100
(plerixafor; Mozobil) which was approved by the FDA for use in
combination with granulocyte-colony stimulating factor for
autologous transplants in NHL and MM patients (Dar et al., 2011).
Another CXCR4 inhibitor, AMD3465, was shown to antagonize CXCL12-
and stroma-induced chemotaxis and inhibited CXCL12-induced
activation of prosurvival signaling pathways in leukemic cells
(Zeng et al., 2009). Further, it was demonstrated that AMD3465,
alone or in combination with granulocyte colony-stimulating factor,
induced mobilization of AML cells and progenitor cells into
circulation and enhanced antileukemic effects of chemotherapy and
sorafenib, resulting in markedly reduced leukemia burden and
prolonged survival of the animals (Zeng et al., 2009). Such
findings suggest that disruption of CXCR4/CXCL12 interactions may
be used to sensitize leukemic cells to chemotherapy by targeting
their protective bone marrow microenvironment.
[0137] As described in the Examples, novel first-in-class human
therapeutic monoclonal antibodies directed to CXCR4 have been
developed. These monoclonal antibodies bind to CXCR4-expressing
cells with low nanomolar affinity, block CXCL12 binding to
CXCR4-expressing cells and inhibit CXCL12-induced migration and
calcium flux with low nanomolar EC.sub.50 values. Significantly, in
addition to blocking CXCL12-induced calcium flux and migration,
data provided in the Examples also indicate that antibody-dependent
induction of apoptosis of CXCR4-expressing tumor cells is a
mechanism of action of these human anti-CXCR4 antibodies.
Antibody-induced apoptosis resulted in robust in vivo efficacy
across multiple hematopoietic tumor xenograft models. Based on the
action of small-molecule CXCR4 antagonists in increasing
mobilization of CXCR4.sup.+ tumor cells from the BM and thereby
increasing chemosensitization, but in not directly killing such
tumor cells, the efficacy of the present anti-CXCR4 antibodies in
killing cancer cells was surprising and unexpected.
[0138] Because CXCR4 plays a role in multiple fundamental aspects
of cancer including proliferation, migration/invasion and
angiogenesis, an antagonist has potentially multiple means to
intervene in malignancies where CXCR4 is expressed. To begin to
dissect the pathway, fully human monoclonal antibodies directed
against CXCR4 and CXCL12, respectively, were developed. Both the
anti-CXCR4 and anti-CXCL12 antibodies inhibit ligand binding to
CXCR4 resulting in inhibition of ligand-induced cellular responses
such as calcium flux and migration (Examples 4-6). In addition to
these functions, the CXCR4/CXCL12 axis has been implicated in
promoting angiogenesis (Guleng et al., 2005); Ping et al., 2011).
Both anti-CXCR4 (Example 7) and anti-CXCL12 (data not shown)
antibodies also inhibited endothelial tube formation, an in vitro
demonstration of angiogenesis.
[0139] To investigate the effects of the disruption of CXCR4/CXCL12
interactions, the efficacy of the antibodies in attenuating tumor
growth was tested in diverse in vivo xenograft models. In a model
for NHL (Burkitt's lymphoma), Ramos cells were engrafted into SCID
mice and rituximab was used as a positive control. Surprisingly,
anti-CXCL12 antibody did not control tumor growth and appeared
indistinguishable from vehicle and isotype control. In contrast,
anti-CXCR4 antibody BMS-936564 demonstrated nearly complete tumor
growth control with similar activity as rituximab (Example 14).
Because in vitro blockade of chemotaxis was similar between the two
antibodies (Example 6), it is unlikely that anti-tumor control is
dependent on blockade of the CXCL12/CXCR4 axis. Consequently, a
direct cytotoxic effect of BMS-936564 was tested in a Ramos cell
proliferation assay. CXCL12 has been implicated as an autocrine
factor promoting cell growth and in a separate study CXCL12 siRNA
inhibited BR5-1 growth (Liu et al., 2011; Righi et al., 2011).
Though the inhibition of growth was partial, a dose-dependent
inhibition of proliferation with anti-CXCR4 was observed, whereas
AMD3100 and anti-CXCL12 antibody had no effect (Example 8).
Recently, a 14-residue polypeptide reported to be a specific CXCR4
antagonist (BKT140) was shown to inhibit proliferation of multiple
myeloma cells (Beider et al., 2011). It has been suggested that
AMD3100 is a weak partial agonist while BKT140 acts as an inverse
agonist (Zhang et al., 2002).
[0140] In view of the foregoing, the anti-CXCR4 antibodies of this
disclosure can be used in a method for treating a subject afflicted
with a CXCR4-expressing cancer comprising administering to the
subject a therapeutically effective amount of an antibody or a
fragment thereof that specifically binds to a CXCR4 receptor
expressed on the surface of a cancer cell. In certain embodiments,
the treatment method is used prophylactically on a subject who was
previously afflicted with, or a subject who is at risk of
contracting, a cancer. In preferred embodiments, the subject is a
human and the antibody or fragment thereof binds to a human CXCR4
receptor. In other preferred embodiments, the antibody or a
fragment thereof that binds to the CXCR4 receptor inhibits the
activity of the receptor. Accordingly, the antibody or fragment
thereof disrupts the homing and maintenance of hematopoietic stem
cells within the BM microenvironment and/or increases mobilization
of cells from the BM to the periphery, and thereby increases the
sensitivity of hematopoietic cancer cells to chemotherapeutic
agents. In other preferred embodiments, the anti-CXCR4 antibody or
fragment thereof induces apoptosis of a CXCR4-expressing cell.
Apoptosis of target cancer cells permits use of the antibody as
monotherapy.
[0141] In certain embodiments, the antibody or fragment thereof is
a chimeric, humanized, or human antibody or a fragment thereof. In
preferred embodiments, the antibody or fragment thereof is a human
antibody or a fragment thereof. In other preferred embodiments, the
antibody or fragment thereof comprises the CDR1, CDR2 and CDR3
domains in a heavy chain variable region comprising consecutively
linked amino acids, the sequence of which is set forth in SEQ ID
NO: 25, and the CDR1, CDR2 and CDR3 domains in a light chain
variable region comprising consecutively linked amino acids, the
sequence of which is set forth in SEQ ID NO: 29.
[0142] In certain embodiments, according to an delineation of CDR
sequences by the Kabat system, the anti-CXCR4 antibody or fragment
thereof comprises a heavy chain variable region CDR1 comprising
consecutively linked amino acids, the sequence of which is set
forth in SEQ ID NO: 1, a heavy chain variable region CDR2
comprising consecutively linked amino acids, the sequence of which
is set forth in SEQ ID NO: 5, a heavy chain variable region CDR3
comprising consecutively linked amino acids, the sequence of which
is set forth in SEQ ID NO: 9, a light chain variable region CDR1
comprising consecutively linked amino acids, the sequence of which
is set forth in SEQ ID NO: 13, a light chain variable region CDR2
comprising consecutively linked amino acids, the sequence of which
is set forth in SEQ ID NO: 17, and a light chain variable region
CDR3 comprising consecutively linked amino acids, the sequence of
which is set forth in SEQ ID NO: 21.
[0143] In other embodiments of the present methods, the anti-CXCR4
antibody or fragment thereof comprises a heavy chain variable
region comprising consecutively linked amino acids having the
sequence set forth in SEQ ID NO: 25 and a light chain variable
region comprising consecutively linked amino acids having the
sequence set forth in SEQ ID NO: 29. In preferred embodiments, the
anti-CXCR4 antibody or fragment thereof is an IgG1 or IgG4 antibody
or a fragment thereof. In more preferred embodiments, the antibody
or fragment thereof is BMS-936564 or a CXCR4-binding fragment
thereof.
[0144] Cancers amenable to the methods of treatment described
herein include solid tumors and hematological malignancies. In
certain embodiments, the solid tumor is selected from breast,
ovarian, prostate, non-small cell lung, pancreatic, thyroid,
colorectal, and kidney cancer, nasopharyngeal carcinoma, melanoma,
renal cell carcinoma, neuroblastoma, glioblastoma,
rhabdomyosarcoma, and osteosarcoma. In other embodiments, the
hematologic malignancy is selected from multiple myeloma, acute
myeloid lymphoma, non-Hodgkin's lymphomas, chronic lymphoid
leukemia, follicular lymphoma, diffuse large B-cell lymphoma,
Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor
B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic
leukemia, mycosis fungoides, anaplastic large cell lymphoma, and
precursor T-lymphoblastic lymphoma. In preferred embodiments, the
hematologic malignancy is multiple myeloma, non-Hodgkin's lymphoma,
diffuse large B-cell lymphoma, follicular lymphoma, acute myeloid
lymphoma, acute lymphoblastic leukemia, or chronic lymphoid
leukemia.
[0145] Multiple myeloma (MM) is a plasma cell malignancy
characterized by the accumulation of malignant, immunoglobulin
secreting, plasma cells within the bone marrow, which can lead to
bone destruction, marrow failure, renal impairment, and peripheral
neuropathy. The median survival after conventional treatments is
3-4 years and can be extended to 5-7 years with high-dose treatment
followed by autologous hematopoietic stem-cell transplantation
(I-ISCT) (Raab et al., 2009).
[0146] Currently approved regimens commonly used for MM include
melphalan-based regimens for induction, and bortezomib
(VELCADE.RTM.) or immunomodulatory drugs (IMiDs) including
thalidomide or lenalidomide (REVLIMID.RTM.)-based regimens for
induction and for subjects in relapse. For subjects with relapsed
or refractory MM, treatment options include I-ISCT, repeat of
previous chemotherapy treatment regimen, or a new regimen. HSCT is
associated with a higher risk of treatment related morbidity.
Furthermore, some subjects are not eligible for I-ISCT, due to poor
performance status or comorbidities. There is currently no cure,
and current therapies can only slow disease progression, prolong
survival, and minimize symptoms. Nearly all MM subjects who survive
their initial therapy relapse or become refractory, regardless of
the line of therapy, and require further therapy (Jemal et al.,
2005). Therefore, there is significant unmet medical need for
subjects with MM. In a preferred embodiment of the present
treatment methods, the hematologic malignancy is multiple myeloma,
including relapsed or refractory MM.
[0147] Acute myeloid leukemia (AML) is the most common acute
leukemia in adults, accounting for 80% of cases. Over 13,000
patients in the U.S. are diagnosed with AML per year, with over
8,820 deaths (Cancer Facts and Figures, 2008). Treatment for adult
AML includes induction chemotherapy to achieve remission and
post-remission chemotherapy (with or without stem cell
transplantation) to avoid relapse. Remission induction rates range
from 50% to 85%. Disease recurs in a majority of subjects.
Treatment of relapsed AML is associated with relatively low
remission rates with few patients deriving durable benefit (Breems
et al., 2005).
[0148] Current options for treating adults with relapsed or
refractory AML include chemotherapy and HSCT. Allogeneic HSCT is
considered the treatment of choice for primary induction failure or
beyond first complete remission (CR) and results in long term
disease-free survival in only about 20% of patients. However, HSCT
is not appropriate or available to a large number of patients for
various reasons (e.g., early relapse, inaccessibility of transplant
facility). This, together with the facts that patients who have
relapsed or are refractory to conventional chemotherapy have poorer
prognoses and responses to chemotherapy compared with those with
newly diagnosed acute leukemia, requires that novel, targeted
agents need to be developed for this patient population. In a
preferred embodiment of the present treatment methods, the
hematologic malignancy is acute myeloid leukemia, including
relapsed AML.
[0149] Chronic lymphocytic leukemia (CLL) is the most common
leukemia in Western countries and accounts for 30% of all leukemias
in the U.S. Approximately 14,570 new cases of CLL will be diagnosed
in 2011 (Siegel et al., 2011), and 4,400 patients will die. The
disease is characterized by a progression of functionally
incompetent, monoclonal lymphocytes, leading to lymphadenopathy,
splenomegaly, hepatomegaly, and a prominent lymphocytosis in the
peripheral blood and bone marrow. Most CLL patients initially
demonstrate a complete or partial remission to chemotherapy, but
with the exception of those treated by HSCT, nearly all relapse
following discontinuation of treatment or develop refractory
disease. Current initial treatment for CLL includes conventional
chemotherapy and/or monoclonal antibody (rituximab) therapy.
Survival for most patients is 5-10 years with increasing morbidity
over time. For patients with relapsed/refractory CLL, the current
treatment options do not cure the disease and there is an estimated
median survival of 16 months. In a preferred embodiment of the
present treatment methods, the hematologic malignancy is chronic
lymphocytic leukemia, including relapsed CLL.
[0150] Follicular lymphoma (FL) is the second most common lymphoma
in the United States and Western Europe, accounting for about 20%
of NHL (overall), and the majority of low-grade lymphomas. Despite
the fact that most patients respond to initial therapy (with about
40-80% complete remission), depending upon the regimen used, nearly
all patients later develop progressive disease. Also, up to 10% are
refractory to their initial treatment. Therefore, new, more
effective therapies are needed. In a preferred embodiment of the
present treatment methods, the hematologic malignancy is follicular
lymphoma, including relapsed FL.
[0151] Diffuse large B-cell lymphoma (DLBCL) is the most common
type of NHL, accounting for 25-30% of adult cases (40% of NHL among
patients more than 75 years old). DLBCL has several subtypes,
including but not limited to germinal center B (GCB) type,
activated B-cell type (ABC) and primary mediastinal (Gisselbrecht
et al., 2011). The 3-year overall survival (OS) for GCB and ABC in
treated patients is 84% and 56%, respectively. Most DLBCL patients
are not cured with conventional therapy. After relapse, while at
least 60% of patients remain sensitive to conventional treatment,
fewer than 10% have prolonged disease-free survival with
second-line treatment regimens (Gisselbrecht et al., 2010).
Relapsed or refractory (r/r) DLBCL is treated with chemotherapy
(with or without rituximab) with the goal of subsequent high-dose
chemotherapy and transplant, for the subset of patients with
chemosensitive disease. Approximately 50% of responders to a second
chemotherapy regimen followed by HSCT maintain their response at 2
years. For non-transplant candidates who fail second line therapy
or who relapse post-transplant, therapy is palliative. Without
transplant, chemotherapy provides short-term disease control in r/r
DLBCL. Primary refractory patients are unlikely to achieve CR with
a second chemotherapy regimen and following relapse, a second
remission is usually not durable (Singer et al., 1986). As DLBCL is
initially a chemoresponsive disease, adding an agent such as an
anti-CXCR4 antibody of this disclosure to restore chemosensitivity
is a sound strategy for treating this disease. In a preferred
embodiment of the present treatment methods, the hematologic
malignancy is diffuse large B-cell lymphoma, including relapsed or
refractory DLBCL.
[0152] Prompted by data from HIV-1 studies showing CXCR4-mediated
apoptosis by binding of HIV-1 envelope glycoprotein-gp120 to CXCR4
(Garg et al., 2006; Berndt et al., 1998), the ability of an
anti-CXCR4 antibody of the disclosure, BMS-936564, to induce
apoptosis of CXCR4-expressing cell lines was measured.
BMS-936564-induced apoptosis was demonstrated in over 20 different
CXCR4-expressing cell lines (see Example 11, and Tables 3 and 4),
confirming that this mechanism is not restricted to one cell
type.
[0153] Apoptosis was also demonstrated in an in vitro model of
minimal residual disease (MRD) for chronic lymphocytic leukemia
(Kashyap et al., 2012). Eradication of MRD is one of the most
challenging goals of treatment of CLL. In this MRD model, which is
based on coculture of stromal cells that express and secrete CXCL12
and provide survival support to primary leukemia cells from CLL
patients, CLL cells exhibited increased viability (20-60% at 48
hours) and showed resistance to chemotherapy agents. However,
nanomolar concentrations (2-200 nM) of BMS-936564 induced cell
death in CLL cells cultured alone as well as those incubated using
the MRD model. The proapoptotic activity of BMS-936564 appeared to
be P53 independent as apoptosis was observed in CLL cells from
patients with 17p deletion and fludarabine resistance in vitro.
BMS-936564 also inhibited CXCL12-mediated F-actin polymerization in
CLL cells at lower concentrations than with AMD-3100, a small
molecule CXCR4 inhibitor. These data suggest that BMS-936564 can
effectively target CLL cells present in the tumor microenvironment
in vivo that may contribute to MRD (Kashyap et al., 2012).
[0154] The apoptotic effect of the disclosed anti-CXCR4 antibodies,
a property not exhibited by small-molecule CXCR4 antagonists, e.g.,
AMD3100, indicates that these antibodies can be used alone, as
monotherapy, to treat patients with cancer. Previous studies on the
effect of CXCR4 antagonists in in vivo AML and MM tumor models have
suggested that these antagonists are effective in enhancing the
sensitivity of the tumors cells to chemotherapy (Azab et al., 2009;
Zeng et al., 2009). In contrast, the data presented herein in the
Examples demonstrate that a statistically significant tumor growth
inhibition was achieved when BMS-936564 was administered as
monotherapy in a wide variety of AML, NHL and MM models.
Accordingly, in certain embodiments of the present treatment
methods, the anti-CXCR4 antibody or fragment thereof is
administered as monotherapy. In preferred embodiments, the antibody
or fragment thereof induces apoptosis of a CXCR4-expressing cell.
Accordingly, this disclosure provides a method of inducing
apoptosis of CXCR4-expressing cancer cells, including cells of the
majority of hematological malignancies, comprising administering to
a subject afflicted with the cancer, a therapeutically effective
amount of an antibody or a fragment thereof that binds specifically
to a CXCR4 receptor expressed on a cell surface.
[0155] Since BMS-936564 is an IgG4 antibody, the in vivo efficacy
cannot be explained by ADCC or CDC. However, it is possible that
the antibody, once bound to CXCR4-expressing cells, engages
Fc.gamma.R1 receptors expressed on antigen presenting cells leading
to phagocytosis. The cell lines, in which BMS-936564 efficacy was
observed in vivo, required a secondary anti-Fc antibody to
BMS-936564 to induce apoptosis in vitro (Example 11). This may be a
consequence of lower expression of CXCR4 on those particular cell
lines. If the mechanism of apoptosis initiation is dependent upon
bringing CXCR4 molecules into close proximity, and the density of
CXCR4 on the cell surface is low relative to the binding distance
spanned by the anti-CXCR4 antibody, then a secondary high-affinity
anti-Fc antibody may be required to bridge that gap, bringing the
receptors together to drive an apoptotic signal. In vivo, this may
be accomplished through Fc.gamma.R1 receptors.
[0156] The data described herein suggest a novel mechanism of
action, involving apoptosis of CXCR4-expressing target cells, for
an anti-CXCR4 antibody in addition to its role in cellular
mobilization. These data indicate that BMS-936564 may provide
effective therapy for hematologic malignancies including MM, AML,
and various NHLs, such as FL and DLBCL, as well as for solid tumor
malignancies. However, the present methods are not necessarily
limited to any particular mechanism of action of the anti-CXCR4
antibodies of the disclosure. For example, CXCR4 may modulate the
epithelial to mesenchymal transition (EMT) in MM cells and the
anti-CXCR4 antibodies of this disclosure may inhibit EMT, as
evidenced by the demonstration that BMS-936564 inhibits the
EMT-related proteins Twist, Snail and Slug, and up-regulates
E-Cadherin (Roccaro et al., 2012). These data corroborate the view
that CXCR4 may represent a valid therapeutical target due to its
ability to modulate EMT.
[0157] It has previously been shown the CXCR4/CXCL12 axis plays a
major role in homing and trafficking of MM cells to the BM, and
disruption of the interaction of tumor cells with the BM leads to
enhanced sensitivity to therapeutic agents (Alsayed et al., 2007;
Azab et al., 2009). These findings suggest that the novel
anti-CXCR4 human antibody, BMS936564, may prevent the homing and
adhesion of MM cells to the BM and sensitize these cells to
therapeutic agents. Notably, the validity of this basis for
targeting CXCR4 is further substantiated by in vivo data reported
by Roccaro et al. (2012) who used primary MM cells (CD138.sup.+),
MM cell lines (MM.1S, RPMI.8226), and primary MM bone marrow
stromal cells (BMSCs) to evaluate migration towards CXCL12 and
BMSCs. Cytotoxicity and DNA synthesis were measured by MTT and
thymidine uptake, respectively. An in vivo melanoma mouse model was
used to validate the effect of anti-CXC4 on modulating tumor cell
metastasis. It was demonstrated that (1) mice treated with
BMS-936564 presented with a less MM cell dissemination to distant
bone marrow niches, compared to vehicle-treated mice, supporting
the hypothesis that CXCR4 may represent a crucial modulation of
tumor cell dissemination; (2) in the melanoma xenograft model
BMS-936564-treated mice exhibited a reduced number of metastasis
compared to vehicle-treated mice; and (3) BMS-936564 functionally
target MM cells in vitro in terms of migration, adhesion and
survival (Roccaro et al., 2012).
[0158] It was further demonstrated that BMS936564 inhibited
migration of MM cells toward CXCL12 and primary MM BMSCs in a
dose-dependent manner. Adhesion of primary MM cells to BMSCs was
also inhibited by BMS936564 in a dose-dependent manner, while also
inducing cytotoxicity on primary BM-derived CD138.sup.+ cells. The
BMS936564 antibody targeted MM cells in the context of BM milieu by
overcoming BMSC-induced proliferation of tumor cells. In addition,
BMS936564 synergistically enhanced bortezomib-induced cytotoxicity
in MM cells (Roccaro et al., 2012). As described in Example 11,
BMS936564-dependent activation of apoptotic pathways in MM cells
was demonstrated by cleavage of caspase-9 and PARP. CXCL12-induced
ERK-, Akt-, and Src-phosphorylation were inhibited by BMS936564 in
a dose-dependent manner. Importantly, as described in Example 16,
BMS936564 inhibited MM cell proliferation in vivo in xenograft
mouse models.
[0159] In total, these data clearly demonstrate that targeting
CXCR-4 on MM cells with an anti-CXCR4 antibody provides an
effective means, probably employing multiple mechanisms, for
treating cancer in general, and MM in particular.
[0160] The anti-CXCR4 antibodies of the disclosure can also be used
in combination other cancer treatments, such as surgery and/or
radiation, and/or can be co-administered with one or other more
therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or
an immunosuppressive agent, which enhances or augments the
therapeutic effect of the anti-CXCR4 antibodies. The antibody can
be linked to the agent (as an immunoconjugate) or can be
administered separate from the agent. In the latter case (separate
administration), the antibody can be administered before, after or
concurrently with the agent or can be co-administered with other
known therapeutic agents, including conventional chemotherapeutic
drugs and antibodies that bind tumor-associated antigens or
immunoregulatory targets. Chemotherapeutic drugs include, among
others, doxorubicin (adriamycin), cisplatin bleomycin sulfate,
carmustine, chlorambucil, cyclophosphamide, lenalidomide,
bortezomib, dexamethasone, mitoxantrone, etoposide, cytarabine,
bendamustine, rituximab, ifosfamide, carboplatin, and etoposide.
Co-administration of an anti-CXCR4 antibody, or antigen binding
fragment thereof, of the present disclosure with chemotherapeutic
agents provides two anti-cancer agents which operate via different
mechanisms which yield a cytotoxic effect to human tumor cells.
Such co-administration can solve problems due to development of
resistance to drugs or a change in the antigenicity of the tumor
cells that would render them unreactive with the antibody.
[0161] In other embodiments, the subject can be additionally
treated with an agent that modulates, e.g., enhances or inhibits,
the expression or activity of Fc.gamma. or Fc.gamma. receptors by,
for example, treating the subject with a cytokine. Preferred
cytokines for administration during treatment with the
multispecific molecule include of granulocyte colony-stimulating
factor (G-CSF), granulocyte-macrophage colony-stimulating factor
(GM-CSF), interferon-.gamma. (IFN-.gamma.), and tumor necrosis
factor (TNF).
[0162] The antibodies of this disclosure also can be used in
combination with one or more additional therapeutic antibodies or
other binding agents, such as Ig fusion proteins. Non-limiting
examples of other antibodies or binding agents with which an
anti-CXCR4 antibody of this disclosure can be administered in
combination include antibodies or binding agents to CTLA-4, PSMA,
CD30, IP-10, IFN-.gamma., CD70, PD-1, PD-L1, KIR, TNF, TNF-R, VEGF,
VEGF-R, CCR5, IL-1, IL-18, IL-18R, CD19, CD52, CS1, EGFR, CD33,
CD20, Her-2, CD25, gpIIb/IIIa, IgE, CD11a, .alpha.4 integrin,
IFN.alpha. and IFNAR1.
[0163] There is growing evidence that disruption of the CXCR4
pathway, and disruption of the interaction between hematological
cancer cells such as MM cells and their bone marrow
microenvironment, confers greater sensitization to anti-cancer
therapies, such as with lenalidomide and bortezomib for MM. As
described in the Examples, nonclinical data on BMS-936564 as
monotherapy and in combination with chemotherapy in MM cell lines
and xenograft studies indicate that BMS-936564 is active in MM and
may enhance the efficacy of regimens such as
lenalidomide/dexamethasone and bortezomib. Preclinical studies have
also shown that CXCR4 inhibition with AMD3100 leads to de-adhesion
of MM cells from bone marrow stromal cells and mobilization of
these cells into the periphery, which results in increased
sensitivity to bortezomib (Azab et al., 2009). The anti-CXCR4
antibodies of the disclosure similarly potentiate the effect of
chemotherapeutics by their ability to release malignant cells from
the protective environment of the BM. In addition to mobilizing MM
cells and increasing their chemosensitization, these antibodies
have the additional effect of directly killing MM cells by
apoptosis (Example 11), among other possible mechanisms. BMS-936564
has been shown to inhibit MM tumor growth in vivo when administered
alone or in combination with lenalidomide or bortezomib (Example
16).
[0164] In certain embodiments of the therapeutic methods described
herein, the method further comprises administering to the subject
at least one chemotherapeutic agent in combination with the
anti-CXCR4 antibody or fragment thereof. In certain embodiments,
the cancer is MM and the at least one chemotherapeutic agent is
lenalidomide plus low-dose dexamethasone, or bortezomib plus
dexamethasone. These chemotherapy combinations are standard
regimens that have proven therapeutic value in subjects with
relapsed or refractory MM, and the safety profile of these
chemotherapy agents is well characterized. In certain preferred
embodiments, the anti-CXCR4 antibody is administered weekly, in
Cycle 1 for the first two weeks as monotherapy, and then in
combination with a chemotherapy regimen that includes lenalidomide
plus low-dose dexamethasone, or bortezomib plus dexamethasone.
[0165] For example, for treatment of MM with BMS-936564 in
combination with lenalidomide and dexamethasone, an exemplary
dosage regimen comprises: (1) BMS-936564 (1, 3, or 10 mg/kg)
administered as a single 60 minute IV infusion on Days 1, 8, 15,
22, 29 and 36 (Cycle 1) and on Days 1, 8, 15, and 22 (Cycle 2 and
subsequent cycles); (2) lenalidomide (25 mg po) administered for 21
days (Days 15-35; Cycle 1) and Days 1-21 (Cycle 2 and subsequent
cycles); and (3) dexamethasone (40 mg) administered on Days 15, 22,
29, and 36 (Cycle 1) and on Days 1, 8, 15, and 22 (Cycle 2 and
subsequent cycles).
[0166] For treatment of MM with BMS-936564 in combination with
bortezomib and dexamethasone, an exemplary dosage regimen
comprises: (1) BMS-936564 (1, 3, or 10 mg/kg) administered as a
single 60 minute IV infusion on Days 1, 8, 15, 22, and 29 (Cycle 1)
and on Days 1, 8, and 15 (Cycle 2 and subsequent cycles); (2)
Bortezomib (1.3 mg/m.sup.2) administered as a 3-5 second IV push on
Days 15, 18, 22, and 25 (Cycle 1) and on Days 1, 4, 8, 11 (Cycle 2
and subsequent cycles); and (3) dexamethasone (20 mg) administered
on Days 15, 16, 18, 19, 22, 23, 25 and 26 (Cycle 1) and on Days 1,
2, 4, 5, 8, 9, 11 and 12 (Cycle 2 and subsequent cycles).
[0167] In certain embodiments, the cancer is AML and the at least
one chemotherapeutic agent administered to a cancer patient in
combination with the anti-CXCR4 antibody or fragment thereof of the
disclosure is mitoxantrone, etoposide, and/or cytarabine, since
this regimen is considered a standard-of-care regimen for relapsed,
refractory AML patients (Amadori et al., 1991). In certain
preferred embodiments, the anti-CXCR4 antibody is administered
weekly, in Cycle 1 for the first two weeks as monotherapy, and then
in combination with a chemotherapy regimen that includes
mitoxantrone, etoposide, and cytarabine.
[0168] For example, for treatment of AML with BMS-936564 as
monotherapy, an exemplary regimen comprises BMS-936564 (0.3, 1, 3,
or 10 mg/kg) administered as a single 60-minute IV infusion on Day
1 in Cycle 1, and on Days 1, 8, and 15 in subsequent Cycles.
[0169] For treatment of AML with BMS-936564 in combination with
chemotherapy, an exemplary dosage regimen comprises: (1) BMS-936564
administered on the first day of chemotherapy prior to the first
dose of chemotherapy. BMS-936564 is administered on Days 1, 8 and
15 in Cycle 2 and subsequent cycles. In addition, for Cycles 2-13,
chemotherapy consists of the following regimen (28-day Cycle): (2)
mitoxantrone (8 mg/m.sup.2 IV) over 15 minutes on Day 1 through 5;
(3) etoposide (100 mg/m.sup.2 IV) over 1 hour on Day 1 through 5;
and (4) cytarabine (Ara-C; 1 g/m.sup.2 IV) over 1 hour on Day 1
through 5.
[0170] In certain embodiments, the cancer is CLL or FL and the at
least one chemotherapeutic agent administered to a cancer patient
in combination with the anti-CXCR4 antibody or fragment thereof of
the disclosure is bendamustine and/or rituximab. Preclinical
studies indicate there is anti-tumor synergy between bendamustine
and rituximab in several leukemia and lymphoma cell lines (Rummel
et al., 2002) such that the latter sensitized B-cell lymphomas to
apoptosis induced by chemotherapies, including bendamustine (Chow
et al., 2002). The bendamustine plus rituximab (BR) combination has
shown efficacy in lymphoma patients who are naive, pretreated or
refractory to rituximab (Friedberg et al., 2008). In certain
preferred embodiments, the anti-CXCR4 antibody is administered in
combination with bendamustine and rituximab.
[0171] In preferred embodiments of a method for treating DLBCL, the
anti-CXCR4 antibody is used in combination with rituximab,
ifosfamide, carboplatin, and/or etoposide (Kewalramani et al.,
2004). No chemotherapy regimen has shown superiority in relapsed or
refractory DLBCL. R-ICE (rituximab, ifosfamide, carboplatin, and
etoposide) is one of the most commonly used regimens in r/r DLBCL
due to its comparable efficacy to other regimens and decreased
toxicity relative to R-DHAP (dexamethasone, high-dose cytarabine,
cisplatin) followed by high-dose chemotherapy and autologous HSCT
for responding patients (Gisselbrecht et al., 2010). In certain
preferred embodiments, the anti-CXCR4 antibody is administered in
combination with rituximab, ifosfamide, carboplatin, and
etoposide.
[0172] For example, for treatment of FL, DLBCL and CLL subjects
with BMS-936564 as monotherapy, a preferred regimen comprises
BMS-936564 (0.3-10 mg/kg) administered as a single 60-minute IV
infusion on Day 1 in Cycle 1, and on Days 1, 8, 15, 22, 29, 36, 43
and 50 in subsequent cycles.
[0173] When administered in combination with chemotherapy for
treatment of CLL, FL, and DLBCL subjects, an exemplary embodiment
comprises administration of BMS-936564 on the first day of
chemotherapy prior to the first dose of chemotherapy, and
administration of chemotherapy at least 1 hour after completion of
the infusion of BMS-936564. BMS-936564 is administered on Days 1
and 8 in Cycle 2 and subsequent Cycles.
[0174] Chemotherapy for CLL consists of the following regimen
(28-day Cycle): rituximab (375 mg/m.sup.2 IV) on Day 1 of Cycle 2
and subsequent cycles, then 500 mg/m.sup.2 on Day 1 of subsequent
cycles; and bendamustine (70 mg/m.sup.2 IV) over 60 minutes on Day
1 of Cycle 2.
[0175] Chemotherapy for FL consists of the following regimen
(28-day Cycle): rituximab (375 mg/m.sup.2 IV) on Day 1 of Cycle 2
and subsequent cycles, then 500 mg/m.sup.2 on Day 1 of subsequent
cycles; and bendamustine (90 mg/m.sup.2 IV) over 60 minutes on Day
1 of Cycle 2.
[0176] Chemotherapy for DLBCL consists of the following regimen
(28-day Cycle): rituximab (375 mg/m.sup.2 IV) on Day 1 of Cycle 2
and subsequent cycles; ifosfamide (5000 mg/m.sup.2) continuous IV
infusion, on Day 4, along with Mesna (2-mercaptoethane sulfonate
Na; 5000 mg/m.sup.2) continuous IV infusion over 24 hr, starting on
Day 4 of Cycle 2 and subsequent cycles; carboplatin (dosage to
yield target AUC 5 mg/mLmin calculated by the Calvert formula;
maximum dose=800 mg), on Day 4 of Cycle 2 and subsequent cycles;
etoposide (100 mg/m.sup.2 IV) daily on Days 3-5 of Cycle 2 and
subsequent cycles.
[0177] One aspect of this invention is the use of any anti-CXCR4
antibody or antigen-binding portion thereof of the disclosure for
the preparation of a medicament for treating a subject afflicted
with a CXCR4.sup.+ cancer. Uses of any anti-CXCR4 antibody or
antigen-binding portion thereof of the disclosure for the
preparation of medicaments are broadly applicable to the full range
of cancers disclosed herein. In preferred embodiments of these
uses, the cancers include hematological malignancies, such as
relapsed or refractory multiple myeloma, relapsed acute myeloid
lymphoma, relapsed chronic lymphocytic leukemia, relapsed
follicular lymphoma or refractory diffuse large B-cell lymphoma.
This disclosure also provides medical uses of any anti-CXCR4
antibody or antigen-binding portion thereof of the disclosure
corresponding to all the embodiments of the methods of treatment
employing an anti-CXCR4 antibody described herein.
[0178] Also within the scope of the present disclosure are kits
comprising any anti-CXCR4 antibody of antigen-binding fragment or
composition thereof of this disclosure and instructions for use.
Accordingly, this disclosure provides a kit for treating a cancer
in a subject, the kit comprising (a) one or more doses of any of
the anti-CXCR4 antibodies or CXCR4-binding fragment thereof of the
disclosure and (b) instructions for using the anti-CXCR4 antibody
or fragment thereof in any of the therapeutic methods described
herein. For example, in certain embodiments the anti-CXCR4 antibody
in the kit comprises the CDR1, CDR2 and CDR3 domains in a heavy
chain variable region having the amino acid sequence set forth in
SEQ ID NO: 25, and the CDR1, CDR2 and CDR3 domains in a light chain
variable region having the amino acid sequence set forth in SEQ ID
NO: 29. In preferred embodiments, the anti-CXCR4 antibody is
BMS-936564. The kit can further contain one or more additional
therapeutic reagents as described herein, such as an
immunosuppressive reagent, a chemotherapeutic agent or a radiotoxic
agent, or one or more additional antibodies that target different
antigens.
[0179] Kits typically include a label indicating the intended use
of the contents of the kit and instructions for use. The term label
includes any writing, or recorded material supplied on or with the
kit, or which otherwise accompanies the kit. In certain embodiments
of a pharmaceutical kit, the anti-CXCR4 antibody may be co-packaged
with other therapeutic agents in unit dosage form.
[0180] B. Viral Infections, Including HIV Infection
[0181] CXCR4 has been shown to be a coreceptor for HIV entry into T
cells and, additionally, certain murine anti-CXCR4 antibodies have
been demonstrated to be able to inhibit entry of HIV isolates into
T cells (see Hou et al., 1998; Carnec et al., 2005). Thus, CXCR4
can be used as a receptor by viruses for entry into the cell and
antibodies to CXCR4 can be used to inhibit cell entry of such
viruses that use CXCR4 as a receptor. CXCR4-mediated apoptosis by
binding of HIV-1 envelope glycoprotein-gp120 to CXCR4 has been
reported (Garg et al., 2006).
[0182] Investigation revealed that antibodies cross-linked to CXCR4
could mimic the cell death observed with gp120-induction (Berndt et
al., 1998), which suggested that the use of anti-chemokine receptor
antibodies to prevent HIV-1 infection might result in efficient and
rapid destruction of the receptor-expressing T-cells. Accordingly,
the human anti-CXCR4 antibodies of this disclosure can be used to
inhibit entry of a virus into a cell, wherein the virus uses CXCR4
as a receptor for cell entry, such that viral infection is
inhibited. In a preferred embodiment, the antibodies are used to
inhibit entry of HIV into T cells, e.g., in the treatment or
prevention of HIV/AIDS. The antibody can be used alone or in
combination with other anti-viral agents, such as anti-retroviral
drugs such as AZT or protease inhibitors.
[0183] C. Inflammatory Conditions
[0184] The CXCR4/CXCL12 pathway has been shown to play a role in a
variety of inflammatory conditions, including but not limited to
inflammatory liver disease (Terada et al., 2003); autoimmune joint
inflammation (Matthys et al., 2001); allergic airway disease
(Gonzalo et al., 2000); and periodontal disease (Hosokawa et al.,
2005).
[0185] Accordingly, the human anti-CXCR4 antibodies of this
disclosure that inhibit binding of CXCL12 to CXCR4 can be used to
inhibit inflammation in inflammatory disorders, including disorders
selected from the group consisting of inflammatory liver disease,
autoimmune joint inflammation, allergic airway disease, periodontal
disease, rheumatoid arthritis, inflammatory bowel disease, systemic
lupus erythematosus, Type I diabetes, inflammatory skin disorders
(e.g., psoriasis, lichen planus), autoimmune thyroid disease,
Sjogren's syndrome, pulmonary inflammation (e.g., chronic
obstructive pulmonary disease, pulmonary sarcoidosis, lymphocytic
alveolitis) and inflammatory kidney disease (e.g., IgA nephropathy,
glomerulonephritis). The antibody can be used alone or in
combination with other anti-inflammatory agents, such as
non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids
(e.g., prednisone, hydrocortisone), methotrexate, COX-2 inhibitors,
TNF antagonists (e.g., etanercept, infliximab, adalimumab) and
immunosuppressants (such as 6-mercaptopurine, azathioprine and
cyclosporine A).
[0186] D. Angiogenesis
[0187] It has been demonstrated that CXCL12 induces
neovascularization through recruitment of CXCR4-expressing
hemangiocytes (Jin et al., 2006). Moreover, blockade of the
CXCR4/CXCL12 pathway can attenuate in vivo tumor growth by
inhibiting angiogenesis in a VEGF-independent manner (Guleng et
al., 2005). Still further, as demonstrated in Example 7, antibodies
of this disclosure are capable of inhibiting capillary tube
formation in vitro. Accordingly, the anti-CXCR4 antibodies of this
disclosure that inhibit binding of CXCL12 to CXCR4 can be used to
inhibit angiogenesis by interfering with the CXCR4/CXCL12 pathway.
Inhibition of angiogenesis can be used, for example, to inhibit
tumor growth or tumor metastasis (regardless of whether the tumor
is CXCR4.sup.+). The antibody can be used alone or in combination
with other anti-angiogenic agents, such as anti-VEGF
antibodies.
[0188] E. Autologous Stem Cell Transplantation
[0189] Peripheral blood stem cells are the preferred source of stem
cells for use in autologous stem cell transplantation, for example
in the treatment of certain hematological malignancies. Collection
of stem cells from the peripheral blood requires mobilization of
CD34.sup.+ stem cells from BM to the peripheral blood. Various
cytokines, chemokines and adhesion molecules have been implicated
in the regulation of this process (reviewed in Gazitt, 2001),
including the interaction of CXCR4 and SDF-1. Moreover, a small
molecule CXCR4 antagonist has been demonstrated to stimulate rapid
mobilization of CD34.sup.+ stem cells from the BM to the periphery
(see, e.g., Devine et al., 2004; Broxmeyer et al., 2005; Flomenberg
et al., 2005). Accordingly, anti-CXCR4 antibodies of this
disclosure that inhibit CXCR4 activity (i.e., antagonist
antibodies) can be used to stimulate mobilization of CD34.sup.+
stem cells from the BM to the peripheral blood to allow for the use
of such stem cells in transplantation (e.g., autologous
transplantation), for example in the treatment of hematological
disorders, such as multiple myeloma and non-Hodgkin's lymphoma. The
antibody can be used alone or in combination with other agents used
to stimulate mobilization of stem cells, such as G-CSF and/or
GM-CSF. Thus, in another embodiment, the invention provides a
method of stimulating mobilization of CD34.sup.+ stem cells from BM
to peripheral blood in a subject, the method comprising
administering to the subject an anti-CXCR4 antibody of the
invention such that mobilization of CD34.sup.+ stem cells from BM
to peripheral blood is stimulated. The method can further comprise
collecting CD34+ stem cells from peripheral blood, such as for use
in autologous stem cell transplantation.
[0190] The present invention is further illustrated by the
following examples, which should not be construed as further
limiting. The contents of all figures and all references, patents
and published patent applications cited throughout this application
are expressly incorporated herein by reference.
Example 1
Generation of Human Monoclonal Antibodies Against CXCR4 and
CXCL12
[0191] Anti-CXCR4 human monoclonal antibodies were generated using
a combination approach in which, first, transgenic transchromosomic
mice expressing human antibody genes (Medarex KM MOUSE.RTM.,
Milpitas, Calif., described in PCT Publication No. WO 02/43478 and
U.S. Pat. No. 7,041,870) were immunized with human
CXCR4-transfected R1610 cells to raise in the mice a repertoire of
human immunoglobulins specific for human CXCR4 and then, second, a
human antibody library was prepared from spleen cells of the mice
and displayed on phage such that the phage were then screened for
expression of variable region fragments having affinity for CXCR4
by panning with human CXCR4 incorporated into magnetic
proteoliposomes (CXCR4-MPL). Variable region fragments of interest
were recloned into a Fab expression vector and the Fab retested for
antigen binding against transfected CXCR4-expressing cells. Fab
clones F7 (since redesignated MDX-1338 or BMS-936564), F9, D1 and
E2 were selected for further analysis. Whole antibodies were
generated from the Fabs using standard molecular biology
techniques. This combination approach is generally described in
U.S. Pat. No. 6,794,132, and is specifically described in detail in
WO 2008/060367.
[0192] To generate the anti-CXCL12 antibody, Medarex KM.RTM.
transgenic mice were immunized with recombinant human CXCL12
(Peprotech, Rocky Hill, N.J.). Spleen lysates were pooled and
processed as described previously (U.S. Pat. No. 6,794,132). Using
proprietary phage display procedures, Biosite generated antibody
fragments (Fab library). Phage which bound to CXCL12 were selected
using biotinylated-CXCL12. Selected antigen-reactive Fabs were
converted to full length IgG4 (S228P) and re-expressed in CHO
cells.
[0193] Isotype control antibody IgG4 containing the S228P hinge
mutation to reduce half-antibody formation (Angal et al., 1993) was
produced at Biologics Discovery California, Sunnyvale, Calif.).
Example 2
Structural Characterization of Human Anti-CXCR4 Monoclonal
Antibodies F7, F9, D1 and E2
[0194] The cDNA sequences encoding the heavy and light chain
variable regions of the F7, F9, D1 and E2 Fab clones, obtained from
phage display library screening as described in Example 1, were
sequenced using standard DNA sequencing techniques.
[0195] The nucleotide and amino acid sequences of the heavy chain
variable region of F7 are shown in FIGS. 1A and 1n SEQ ID NO: 33
and 25, respectively. The nucleotide and amino acid sequences of
the light chain variable region of F7 are shown in FIGS. 1B and 1n
SEQ ID NO: 37 and 29, respectively.
[0196] Comparison of the F7 heavy chain immunoglobulin sequence to
the known human germline immunoglobulin heavy chain sequences
demonstrated that the F7 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 3-48, a D segment from the human
germline 4-23, and a JH segment from human germline JH 6B. Further
analysis of the F7 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CD3 regions as shown in FIGS. 1A and 1n SEQ ID NOs:
1, 5 and 9, respectively. Comparison of the F7 light chain
immunoglobulin sequence to the known human germline immunoglobulin
light chain sequences demonstrated that the F7 light chain utilizes
a V.sub.L segment from human germline V.sub.K L15 and a JK segment
from human germline JK 1. Further analysis of the F7 V.sub.L
sequence using the Kabat system of CDR region determination led to
the delineation of the light chain CDR1, CDR2 and CD3 regions as
shown in FIGS. 1B and 1n SEQ ID NOs: 13, 17 and 21,
respectively.
[0197] The nucleotide and amino acid sequences of the heavy chain
variable region of F9 are shown in SEQ ID NO: 34 and 26,
respectively. The nucleotide and amino acid sequences of the light
chain variable region of F9 are shown in SEQ ID NO: 38 and 30,
respectively. Comparison of the F9 heavy chain immunoglobulin
sequence to the known human germline immunoglobulin heavy chain
sequences demonstrated that the F9 heavy chain utilizes a V.sub.H
segment from human germline V.sub.H 3-48, a D segment from the
human germline 4-23, and a JH segment from human germline JH 6B.
Further analysis of the F9 V.sub.H sequence using the Kabat system
of CDR region determination led to the delineation of the heavy
chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 2, 6 and
10, respectively. Comparison of the F9 light chain immunoglobulin
sequence to the known human germline immunoglobulin light chain
sequences demonstrated that the F9 light chain utilizes a V.sub.L
segment from human germline V.sub.K L15 and a JK segment from human
germline JK 1. Further analysis of the F9 V.sub.L sequence using
the Kabat system of CDR region determination led to the delineation
of the light chain CDR1, CDR2 and CD3 regions as shown in SEQ ID
NOs: 14, 18 and 22, respectively.
[0198] The nucleotide and amino acid sequences of the heavy chain
variable region of D1 are shown in SEQ ID NO: 35 and 27,
respectively. The nucleotide and amino acid sequences of the light
chain variable region of D1 are shown in SEQ ID NO: 39 and 31,
respectively. Comparison of the D1 heavy chain immunoglobulin
sequence to the known human germline immunoglobulin heavy chain
sequences demonstrated that the D1 heavy chain utilizes a V.sub.H
segment from human germline V.sub.H 3-48, a D segment from the
human germline 4-23, and a JH segment from human germline JH 6B.
Further analysis of the D1 V.sub.H sequence using the Kabat system
of CDR region determination led to the delineation of the heavy
chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs. 3, 7 and
11, respectively. Comparison of the D1 light chain immunoglobulin
sequence to the known human germline immunoglobulin light chain
sequences demonstrated that the D1 light chain utilizes a V.sub.L
segment from human germline V.sub.K L15 and a JK segment from human
germline JK 1. Further analysis of the D1 V.sub.L sequence using
the Kabat system of CDR region determination led to the delineation
of the light chain CDR1, CDR2 and CD3 regions as shown in SEQ ID
NOs. 15, 19 and 23, respectively.
[0199] The nucleotide and amino acid sequences of the heavy chain
variable region of E2 are shown in SEQ ID NO: 36 and 28,
respectively. The nucleotide and amino acid sequences of the light
chain variable region of E2 are shown in SEQ ID NO: 40 and 32,
respectively. Comparison of the E2 heavy chain immunoglobulin
sequence to the known human germline immunoglobulin heavy chain
sequences demonstrated that the E2 heavy chain utilizes a V.sub.H
segment from human germline V.sub.H 3-48, a D segment from the
human germline 4-23, and a JH segment from human germline JH 6B.
Further analysis of the E2 V.sub.H sequence using the Kabat system
of CDR region determination led to the delineation of the heavy
chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 4, 8 and
12, respectively. Comparison of the E2 light chain immunoglobulin
sequence to the known human germline immunoglobulin light chain
sequences demonstrated that the E2 light chain utilizes a V.sub.L
segment from human germline V.sub.K L15 and a JK segment from human
germline JK 1. Further analysis of the E2 V.sub.L sequence using
the Kabat system of CDR region determination led to the delineation
of the light chain CDR1, CDR2 and CD3 regions as shown in SEQ ID
NOs: 16, 20 and 24, respectively.
[0200] Analysis of the framework sequences of the V.sub.H and
V.sub.L regions of F7, F9, D1 and E2, as compared to the germline
sequences from which they were derived, identified various
framework amino acid residues that differed from germline. Certain
framework residues in the N-terminal regions of the V.sub.H and
V.sub.L segments were chosen for "back-mutation" to restore the
framework residue to the germline sequence, because these
non-germline residues in the N-terminal portion were encoded by the
primers used to create the phage display libraries described in
Example 1. In particular, the modified forms of the V.sub.H and
V.sub.L segments of F7, F9, D1 and E2 (referred to as "GL" forms,
for germline) were created using standard molecular biology
techniques to substitute the germline amino acid residue at the
indicated framework position. The specifically back-mutated amino
acids, and alignments of the sequences of the GL variants with the
sequences of the original variable regions of F7, F9, D1 and E2 are
provided in WO 2008/060367.
[0201] The F7, F9, D1 and E2 Fab fragments are converted to
full-length antibodies using standard recombinant DNA techniques.
For example, DNA encoding the V.sub.H and V.sub.K regions of one of
the Fab fragments can be cloned into an expression vector that
carries the heavy and light chain constant regions such that the
variable regions are operatively linked to the constant regions.
Alternatively, separate vectors can be used for expression of the
full-length heavy chain and the full-length light chain.
Non-limiting examples of expression vectors suitable for use in
creating full-length antibodies include the pIE vectors described
in U.S. Pat. No. 7,674,618. The F7 (BMS-936564) Fab fragments were
converted to a full-length IgG4 (S228P) antibody and re-expressed
in CHO cells.
Example 3
Binding Characteristics of Anti-CXCR4 Human Monoclonal
Antibodies
[0202] In this example, binding characteristics of the anti-CXCR4
antibodies were examined by flow cytometry.
[0203] The human T cell line CEM, which expresses native human
CXCR4 on its cell surface, was used to examine the ability of the
F7, F9, D1 and E2 antibodies to bind to native, cell-surface CXCR4.
Full-length F7, F9, D1 and E2 were titrated in a 1:3 serial
dilution series, resulting in a concentration range from 300 nM to
5 pM. The antibodies were then mixed with CEM cells and allowed to
bind before being detected with a FITC-conjugated anti-human IgG
secondary antibody. The cells were then analyzed by fluorescence
cytometry. The resulting mean fluorescence intensities are shown in
the graph of FIG. 2, which demonstrates that all four anti-CXCR4
antibodies bind to CEM cells. The EC.sub.50 for binding F7, F9, D1
and E2 were 21 nM, 14 nM, 80 nM and 290 nM, respectively.
[0204] To determine the ability of a panel of anti-CXCR4 antibodies
to compete for binding to CXCR4, competition studies were
performed. The four human anti-CXCR4 antibodies F9, F7, E2 and D1
were used, along with four commercially available murine monoclonal
anti-CXCR4 antibodies (12G5, 708, 716 and 717; R&D Systems
catalog Nos. MAB170, MAB171, MAB172 and MAB173, respectively). The
anti-CXCR4 antibodies were titrated in a 1:3 serial dilution series
resulting in a concentration range from 300 nM to 5 pM in the
presence of a constant concentration of FITC-labeled anti-CXCR4
antibody F9. The mixture of antibodies was then added to CEM cells
and allowed to bind. The ability of each antibody to compete with
F9 for binding to CEM cells was assessed by fluorescent cytometry
and detection of FITC. The resulting mean fluorescent intensities
are shown in the graph of FIG. 3, which demonstrates that all seven
antibodies examined (F7, E2, D1, 12G5, 708, 716 and 717) were able
to compete with F9 for binding to CEM cells, although the E2
antibody only demonstrated partial inhibition at high
concentrations compared to the other antibodies.
[0205] In another set of experiments, the ability of the BMS-936564
mAb to bind to a variety of different cell lines was examined by
flow cytometry by carrying out an FACS titration. Increasing
amounts of mAb (from less than 0.001 .mu.g/ml to more than 100
.mu.g/ml) were incubated with 100,000 cells and binding assessed by
flow cytometry. The Bmax value also was determined, which indicates
approximately how many CXCR4 molecules are present on each cell.
Based on the binding curves, an EC.sub.50 for antibody binding was
determined, the results of which are summarized below in Table
1.
TABLE-US-00001 TABLE 1 FACS Titration Results for F7 (BMS-936564)
Binding to Different Cell Lines Cell Type EC.sub.50 (.mu.g/ml) Bmax
Ramos 0.48 106,000 Raji 0.34 52,536 Namalwa 1.57 116,000 L540 3.69
31,868 DMS79 3.99 24,587 MDA-MB-231 9.24 14,186 Bmax = maximum
binding (GMFI units)
[0206] The results show that the F7 mAb (BMS-936564) is capable of
binding effectively to each of the six cell lines tested, with the
lowest EC.sub.50's observed with the Ramos and Raji cell lines.
These data also show that the expression of CXCR4 receptor is
highest for Ramos and Namalwa cells and lowest for MDA-MB-231 cells
and DMS79 cells.
[0207] In another binding experiment, the ability of the BMS-936564
mAb to bind to different subsets of human peripheral blood
mononuclear cells (PBMCs) was examined. Human PBMCs were isolated
by standard methods and different cellular subsets were isolated by
FACS. In particular, the following cellular subsets were isolated:
(i) CD3.sup.+, (ii) CD20.sup.+; (iii) CD11b.sup.+ and (iv)
CD14.sup.+. Flow cytometry experiments conducted with the
BMS-936564 mAb (at 33 .mu.g/ml) demonstrated that it was capable of
binding effectively to each of the four subsets, as compared to an
isotype-matched control antibody.
[0208] In another experiment, a different set of human CXCR4.sup.+
cell lines were evaluated for BMS-936564 binding using flow
cytometry. Cells were prepared for flow cytometry (FACS) staining
by suspending cells with the indicated concentrations of naked
BMS-936564 or biotinylated BMS-936564 before incubating the mixture
of antibody and cells with goat anti-human FC.gamma.-PE or
PE-conjugated streptavidin. Cells were analyzed by FACS by gating
on the live cell population identified by FSC and SSC.
Dose-dependent binding was seen for the cell lines R1610-huCXCR4
(R1610 hamster fibroblasts transfected with human CXCR4 and kept
under G418 selection); Ramos (human B lymphoblast Burkitt's
lymphoma); CEM (human T lymphoblast acute lymphoblastic leukemia);
Nomo-1 (human acute myeloid leukemia); HL-60 (human promyeloblast);
MOLP8 (human MM); and JJN-3R (human MM cell line selected for
resistance to bortezomib). See FIG. 4. No binding to the R1610
parental cells was detected. Based upon geometric mean fluorescent
intensity (GMFI), CXCR4 levels were highest on R1610-huCXCR4 and
Ramos cells followed by CEM (FIG. 4B), Nomo-1 and HL60 (FIG. 4A).
The multiple myeloma cell lines MOLP-8 and JJN-3R expressed the
lowest number of receptors (FIG. 4C). The EC.sub.50 values for
binding are shown in Table 2. In addition, BMS-936564 bound to
healthy donor PBMCs (data not shown) as well as 7/8 PBMCs samples
collected from AML patients with variable GMFI (FIG. 4D). These
data indicate that CXCR4 is expressed on multiple hematopoietic
cell lines and variably expressed in AML patients.
TABLE-US-00002 TABLE 2 Binding of BMS-936564 Binding to Human
CXCR4.sup.+ Cell Lines Cell Type EC.sub.50 (nM) R1610-huCXCR4 2.3
Ramos 4.2 CEM 10.3 Nomo-1 40 HL-60 5.3 MOLP-8 6.5 JJN-3R 2.0
Example 4
Inhibition of CXCL12 Binding to CXCR4 by Anti-CXCR4 and Anti-CXCL12
Antibodies
[0209] To determine the ability of the anti-CXCR4 human antibodies
to inhibit the binding of CXCL12 to CXCR4, a competition study was
performed using .sup.125I-labeled CXCL12 (PerkinElmer, Waltham,
Mass.) and CEM cells, which naturally express CXCR4. A comparison
of anti-CXCR4 antibodies on blocking CXCL12 binding to CEM cells
was performed by a standard radio-labeled ligand binding assay. The
anti-CXCR4 antibodies were serially diluted 1:3 to yield a range of
concentrations from 300 nM to 137 pM. The antibodies were added to
750,000 CEM cells in 100 .mu.l in the presence of 100 pM
.sup.125I-CXCL12 with a specific activity of 2000 Ci/mmole
(Amersham, catalog #IM314-25UCI). An irrelevant antibody of the
same isotype was used as a negative control. The total possible
bound radio-labeled ligand was determined by allowing the
.sup.125I-CXCL12 to bind to CEM cells in the absence of antibodies
for 2 hours at 4.degree. C. Non-specific binding of the
radio-labeled ligand was determined by allowing the
.sup.125I-CXCL12 to bind in the presence of 1 .mu.M unlabeled
CXCL12 (Peprotech, catalog #300-28A). The amount of cell-associated
.sup.125I-CXCL12 was determined by standard methods. The results
are shown in FIG. 5, which demonstrates that the F7 antibody
(BMS-936564) provides the most effective blockade of CXCL12 binding
to CXCR4 expressed on CEM cells. The F9 and D1 antibodies also
blocked CXCL12 binding, although more moderately than F7. The E2
antibody, although it does bind to CXCR4 on CEM cells (as
demonstrated in Example 3), did not effectively block CXCL12
binding to CXCR4 on CEM cells. The EC.sub.50's for CXCL12 blockade
by F7, F9 and D1 were 2.3 nM, 12.5 nM and 28.6 nM,
respectively.
[0210] In another experiment, the blockade of binding of CXCL12 to
CXCR4 by BMS-936564 and an anti-CXCL12 antibody was compared.
Serial dilutions of BMS-936564, anti-CXCL12 and control antibody
were tested for blockade of .sup.125I-CXCL12 binding to CXCR4.sup.+
CEM cells. Competition of .sup.125I-CXCL12 (PerkinElmer, Waltham,
Mass.) binding to CXCR4 on CEM cells was demonstrated using a fixed
concentration of .sup.125I-CXCL12 (100 pM) and a titration of
BMS-936564 from 5 pM to 300 nM. An isotype antibody was used as a
negative control and unlabeled CXCL12 was used as a positive
control. Plates were incubated at room temperature for 1 hour, the
filters were washed, removed and counts per minute (CPM) were read
by a PerkinElmer WIZARD.RTM. gamma counter (Waltham, Mass.). For
all in vitro studies, the data were graphed and analyzed with
GraphPad Prism software (San Diego, Calif.), using nonlinear
regression and sigmoidal dose-response curves.
[0211] Saturation binding studies were conducted using radiolabeled
CXCL12 and CXCR4.sup.hi CEM cells. The K.sub.D of .sup.125I-CXCL12
binding to CEM cells was determined to be 4.3 nM (data not shown)
which is similar to the reported K.sub.D of CXCL12 for CXCR4
ranging from 3.0 to 5.4 nM (DiSalvo et al., 2000). Using a
suboptimal fixed concentration of .sup.125I-CXCL12 (100 pM),
BMS-936564 was titrated and dose-dependent inhibition of
.sup.125I-CXCL12 binding with an EC.sub.50 value of approximately 2
nM was observed (FIG. 6A). Interestingly, the anti-CXCL12 antibody
was more potent and induced a dose-dependent inhibition of
.sup.125I-CXCL12 binding to CEM cells with an EC.sub.50 value of
approximately 90 pM (FIG. 6B).
[0212] BMS-936564 was also shown to block .sup.125I-CXCL12 binding
to Ramos cells in a dose-dependent manner with an EC.sub.50 value
of approximately 11 nM (FIG. 6C).
Example 5
Inhibition of CXCL12-Induced Calcium Flux by Anti-CXCR4 and
Anti-CXCL12 Antibodies
[0213] To determine the ability of the anti-CXCR4 human antibodies
to inhibit calcium flux in CEM cells induced by CXCL12, CEM cells
were first labeled with the fluorescent dye Calcium 3 (Molecular
Devices, Sunnyvale, Calif.). The anti-CXCR4 antibodies were
titrated in a 1:3 serial dilution series resulting in a
concentration range from 100 nM to 1 pM and allowed to bind to
200,000 CEM cells in 200 .mu.l and incubated 10 minutes at room
temperature prior to loading into a FLEXSTATION.RTM. machine
(Molecular Devices). As a negative control, an irrelevant antibody
of the same isotype was used. Cells were then stimulated with a
final concentration of 50 nM recombinant human CXCL12.alpha.
(Peprotech), added as 500 nM in a volume of 22 .mu.l for a final
volume of 222 .mu.l. The resulting calcium flux was measured for
200 seconds per well. As a positive control, cells in the absence
of antibody were stimulated with CXCL12.alpha. (made up in Hank's
buffered saline (FIBS) with 0.1% BSA or HBS) to achieve a maximum
possible calcium flux signal. To determine a baseline, cells were
stimulated with FIBS with 0.1% BSA. The CXCL12.alpha.-stimulated
release of calcium was measured by the development of
calcium-dependent fluorescence over time. The area under the curve
of the resulting fluorescence trace was reported as an indication
of calcium flux. The resulting inhibition of calcium flux by the
anti-CXCR4 antibodies is represented in FIG. 7. The data were
plotted and the EC.sub.50s were calculated using GraphPad Prism
software and the non-linear curve fit, sigmoidal dose response
formula. Antibodies F7 (BMS-936564), F9 and D1 inhibited
CXCL12.alpha.-induced calcium flux. Although antibody E2 did bind
to CXCR4 (as demonstrated in Example 3), it did not significantly
inhibit CXCL12.alpha.-induced calcium flux. The EC.sub.50's for
inhibition of CXCL12-induced calcium flux by F7, F9 and D1 were
0.90 nM, 0.32 nM and 0.57 nM, respectively.
[0214] In another experiment, the capacity of BMS-936564 and
anti-CXCL12 to inhibit CXCL12-induced calcium flux was compared.
Ramos and CELL cells were loaded with FLIPR.RTM. Calcium 4 dye
(Molecular Devices), and a fixed concentration of CXCL12 was used
to stimulate calcium flux. A titration of BMS-936564 or anti-CXCL12
from 50 pM to 100 nM was used to inhibit the response. A maximal
calcium response was set with CXCL12 minus antibodies. A baseline
response was established with buffer stimulation of cells without
CXCL12. Calcium fluxes were read on the FLEXSTATION.RTM. (Molecular
Devices). CXCL12 was shown to induce a dose-dependent rise in
intracellular calcium with peak calcium flux reached at 50 nM and 5
nM with Ramos and CEM cells, respectively. Using the optimal
concentration of CXCL12 to stimulate calcium flux, a titration of
BMS-936564 or anti-CXCL12 was used to inhibit the response (FIGS.
6C and 6D). Both BMS-936564 and anti-CXCL12 blocked CXCL12-induced
calcium flux in a dose dependent manner. BMS-936564 blocked with an
EC.sub.50 of approximately 10 nM and 8 nM in Ramos and CEM,
respectively (FIGS. 8A and 8B), whereas anti-CXCL12 blocked with an
EC.sub.50 of approximately 35 nM (Ramos) and 2 nM (CEM) cells
(FIGS. 8A and 8B).
Example 6
Inhibition of CXCL12-Induced Migration of CEM Cells by Anti-CXCR4
and Anti-CXCL12 Antibodies
[0215] To determine the ability of the anti-CXCR4 human antibodies
to inhibit migration of CEM cells induced by CXCL12, CEM cells
first were labeled with the BATDA
(bis(acetoxymethyl)2,2':6',2''-terpyridine-6,6''-dicarboxylate)
chemiluminescent migration reagent (PerkinElmer). The anti-CXCR4
antibodies were titrated in a 1:3 serial dilution series resulting
in a concentration range from 100 nM to 1 pM and allowed to bind to
labeled CEM cells at a density of 10 million cells per ml. As a
negative control, an irrelevant antibody of the same isotype was
used. Recombinant human CXCL12.alpha. (Peprotech) was added at 5 nM
at 30 .mu.l per well to the lower chamber of a 96 well Neuroprobe
migration plate with 5.7 mm diameter filters per well. Each well
contains 5 .mu.M pores. Labeled CEM cells with and without antibody
were loaded onto the filters at a concentration of 0.5 million
cells per well in a volume of 50 .mu.l. The migration plate was
incubated at 37.degree. C. for 2.5 hours. Migrated cells were
captured in the lower chamber of the plate, lysed and detected with
DELFIA.RTM. Europium detection solution (Perkin Elmer). The
chemiluminescent signal was recorded on a Fusion instrument. The
resulting inhibition of CXCL12.alpha.-induced migration by the
anti-CXCR4 antibodies is shown in FIG. 9. The results demonstrated
that antibodies F7 and F9 inhibited migration effectively, while
antibodies D1 and E2 did not significantly inhibit migration. The
EC.sub.50's for inhibition of CXCL12-induced CEM cell migration by
F7 and F9 were 12.44 nM and 18.99 nM, respectively.
[0216] In another experiment, the ability of BMS-936564 and
anti-CXCL12 to inhibit CXCL12-induced migration of Ramos and CEM
cells was compared. Cells were loaded with BATDA. A fixed
concentration of CXCL12 was used to stimulate migration of cells
through a filter containing 5 .mu.m pores on Migration Plates from
Neuro Probe (Gaithersburg, Md.). A titration of BMS-936564 or
anti-CXCL12 from 20 pM to 300 nM was added to the cells. CXCL12
without antibody was used to establish maximal migration. Migration
toward media alone without CXCL12 was used to measure background
migration. Following a 2-hour incubation at 37.degree. C., migrated
cells were detected by addition of DELFIA.RTM. Europium solution
(Perkin Elmer) to the lysed cells and detected by time resolved
fluorescence on the Fusion instrument. The optimal concentration of
CXCL12 for inducing Ramos migration was established to be 10 ng/mL
(1.25 nM) while CEM cells were more sensitive to CXCL12 and
exhibited maximal migration at 0.05 nM CXCL12. BMS-936564 was shown
to block CXCL12-induced migration with an approximate EC.sub.50
value of 1 nM in Ramos cells and 4 nM in CEM cells (FIGS. 10A and
10B). Anti-CXCL12 inhibited CXCL12-induced migration with an
approximate EC50 value of 0.9 nM (Ramos) and 0.13 nM (CEM) cells
(FIGS. 10A and 10B).
Example 7
Inhibition of HuVEC Capillary Tube Formation by Anti-CXCR4
Antibodies
[0217] In this example, the ability of the anti-CXCR4 human
antibodies to inhibit capillary tube formation by human umbilical
vein endothelial cells (HuVEC) was examined. MATRIGEL.RTM. was
diluted 1:1 with RPMI and plated onto the wells of a 96 well plate
and allowed to polymerize for 30 minutes at 37.degree. C. HuVEC
(from Cambrex, cat. # CC-2519) at 80% confluence were trypsinized
and resuspended at 1.times.10.sup.6 cells per ml in RPMI with 0.5%
FBS. Antibodies were well mixed with HuVEC at a final concentration
of 3 .mu.g/ml and allowed to incubate at room temperature for 30
minutes. An irrelevant antibody of the same isotype or media alone
was used as a negative control. As a positive control of inhibition
of tube formation, a mouse anti-human .alpha.v.beta.3 (CD51/CD61)
antibody (R&D Systems, cat. # MAB3050) was used. HuVEC with or
without antibodies were plated onto the MATRIGEL.RTM.-coated wells
and incubated at 37.degree. C. for 18 hours.
[0218] The HuVEC incubated with media alone or with the
isotype-matched control antibody formed capillary tubes resulting
in the appearance of connected cells across the plate with 3-5
points of connection or branch points per cell. The HuVEC incubated
with either the anti-CXCR4 human antibodies or the
anti-.alpha.v.beta.3 antibody did not form capillary tubes. The
cells appeared isolated and with few or no branch points. The
anti-CXCR4 antibodies that were most effective in blocking CXCL12
binding, CXCL12-induced calcium flux and CXCL12-induced migration,
namely F7 and F9, were also the most effective in inhibiting
capillary tube formation. The anti-CXCR4 antibody E2, which binds
to CXCR4 but does not block CXCL12 binding or CXCL12-induced
effects, did not inhibit capillary tube formation.
Example 8
Anti-CXCR4 Antibodies, but not Anti-CXCL12, Inhibit In Vitro
Proliferation of CXCR4-Expressing Cells
[0219] In this example, the ability of the anti-CXCR4 human
antibodies to inhibit proliferation of Ramos tumor cells (a human
Burkitt's lymphoma cell line) in vitro was examined. In the assay,
1.times.10.sup.4 cells/well were incubated with increasing doses
(10.sup.-3 to 300 nM) of F7 IgG4 antibody, F9 IgG1 antibody, E2
IgG1 antibody, F9 Fab' antibody or isotype controls. The cells were
incubated with antibody for 72 hours, with .sup.3H-thymidine being
added for the final 24 hours of incubation to allow for monitoring
of cell proliferation. Following the incubation, incorporation of
.sup.3H-thymidine by the cells was measured by standard techniques.
The results are shown in the graph of FIG. 11A. The results
demonstrate that the F7 IgG4, F9 IgG1 and E2 IgG1 antibodies each
were able to inhibit Ramos cell proliferation, as indicated by
decreased .sup.3H-thymidine incorporation when incubated with these
antibodies, whereas the F9 Fab' fragment did not inhibit cell
proliferation. These results indicate that the anti-CXCR4 human
antibodies have a direct anti-proliferative effect on the tumor
cells in vitro and thus do not require secondary cross-linking to
achieve an anti-proliferative effect.
[0220] In another experiment, the effects of MDX-1338, anti-CXCL12,
and small-molecule CXCR4 antagonists, AMD3100 and BKT140, on
proliferation of Ramos cells were compared. Ramos cells were
suspended at 1.times.10.sup.5 cells/mL in growth media, incubated
with the relevant antibodies, including isotype controls, and other
test agents and cultured for 72 hours at 37.degree. C.
Cell-Titer-Glo (Promega) was added to wells, mixed, and incubated
at room temperature for 10 minutes. The plate was read on a GloMax
Luminometer (Promega). The results are shown in FIG. 11B. A maximum
of about 50% inhibition of Ramos cell proliferation was seen with
40 nM BMS-936564 treatment compared to isotype control, but
anti-CXCL12 did not inhibit cell proliferation. In addition,
AMD3100, a small molecule CXCR4 antagonist did not inhibit
proliferation. A recently described 14-residue peptide antagonist,
BKT140, did inhibit proliferation but at much higher concentrations
(100 .mu.M). Camptothecin (CPT) completely inhibited cell
proliferation at 10 .mu.M.
Example 9
Inhibition of Solid Tumor Cell Proliferation In Vivo by Anti-CXCR4
Antibodies
[0221] In this example, the ability of the anti-CXCR4 human
antibodies to inhibit proliferation of an established solid tumor
in vivo was examined using a Ramos subcutaneous tumor cell model.
In this assay, 10.times.10.sup.6 Ramos cells/mouse were implanted
into the flank region of each mouse and allowed to grow to a mean
size of 40 mm.sup.3, calculated by
length.times.width.times.height/2 of the tumors. The mice then
received an intraperitoneal (IP) injection of a first dose of
antibody (designated as day 0 of treatment) and received a second
IP dose of antibody on day 7. Mice treated with a Fab' fragment
antibody also received IP antibody doses on day 3 and day 10.
Groups of mice (n=8) were treated with either (i) vehicle; (ii)
isotype control (15 mg/kg); (iii) F7 IgG4 (15 mg/kg); (iv) F9 IgG1
(15 mg/kg); (v) F9 Fab' (10 mg/kg); or (vi) anti-CD20 positive
control (15 mg/kg). Tumor volume and mouse body weight were
measured at regular intervals (approximately 2-3 times/week)
between day 0 and day 30 post dosing. The results of the experiment
are presented in FIGS. 12A, 12B and 12C, which show mean tumor
volume (FIG. 12A), median tumor volume (FIG. 12B) and median % body
weight change (FIG. 12C). The results demonstrated that, like the
positive control, the F7 IgG4 and F9 IgG1 antibodies significantly
inhibited tumor cell growth as measured by increased tumor volume,
whereas the F9 Fab' fragment did not inhibit tumor cell growth as
compared to the isotype control. All treatments were well-tolerated
as indicated by no significant body weight change. The differences
in body weights between treatments were most likely due to the
weights of the tumors. The results indicate that the anti-CXCR4
human antibodies are capable of inhibiting growth of an established
solid tumor i