a new anti-cxcr4 antibody that blocks the cxcr4/sdf-1 axis and … · 2016. 6. 11. · 1 title a...
TRANSCRIPT
1
Title A new anti-CXCR4 antibody that blocks the CXCR4/SDF-1 axis and mobilizes effector cells Running Title A new anti-CXCR4 antibody Authors Matthieu Broussas,1 Nicolas Boute,2 Barbara Akla,1 Sven Berger,2 Charlotte Beau-Larvor,1 Thierry Champion,3 Alain Robert,2 Alain Beck,3 Jean-François Haeuw,4 Liliane Goetsch,1 Christian Bailly,5 Charles Dumontet,6 Thomas Matthes,7 Nathalie Corvaia,8 and Christine Klinguer-Hamour3 Affiliations 1Department of Experimental Oncology, Centre d’Immunologie Pierre Fabre (CIPF), Saint-Julien-en-Genevois, France; 2Unit of Molecular and Cellular Biology, CIPF, Saint-Julien-en-Genevois, France; 3Department of Physico-chemistry, CIPF, Saint-Julien-en-Genevois, France; 4Department of Biochemistry, CIPF, Saint-Julien-en-Genevois, France; 5CDMO Director, Toulouse, France, 6 INSERM 1052/CNRS 5286/University of Lyon, Lyon, France; 7Hematology Service and Service of Clinical Pathology, University Hospital Geneva, Switzerland, and 8Director of the CIPF, Saint-Julien-en-Genevois, France Corresponding author : Christine Klinguer-Hamour. Centre d’Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien en Genevois, France. Tel.: +33 4 50 35 35 83 Fax: + 33 4 50 35 35 90 e-mail: [email protected] Financial support None of the authors received any grant Conflict-of-interest disclosure: All the authors are currently employees of the Institut de Recherche Pierre Fabre excepted, C. Dumontet and T. Matthes. C. Dumontet received research funding from P Fabre for animal experiments. T. Matthes is a Consultant (Advisory board) for Phi Pharma SA (Sion; Switzerland) Keywords: monoclonal antibody, CXCR4, SDF-1, effector functions, signaling pathway. Article category: Research Article. Word count: 5110 (main text + figure captions). Total number of figures: 6 Total number of tables: 1
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
2
Abstract
The type-4 C-X-C-motif chemokine receptor (CXCR4) is expressed in a large variety
of human cancers including hematological malignancies, and this receptor and its ligand,
stromal-cell-derived factor-1 (SDF-1), play a crucial role in cancer progression. We
generated a humanized immunoglobulin-G1 monoclonal antibody, hz515H7, which binds
human CXCR4, efficiently competes for SDF-1 binding, and induces a conformational
change in CXCR4 homodimers. Furthermore, it inhibits both CXCR4 receptor-mediated G-
protein activation and β-arrestin-2 recruitment following CXCR4 activation. The binding of the
hz515H7 antibody to CXCR4 inhibits the SDF-1-induced signaling pathway, resulting in
reduced phosphorylation of downstream effectors such as Akt, Erk1/2, p38 and GSK3β.
Hz515H7 also strongly inhibits cell migration and proliferation and, while preserving normal
blood cells, induces both antibody-dependent cellular cytotoxicity and complement-
dependent cytotoxicity against neoplastic cells. In mouse xenograft models, hz515H7
displays anti-tumor activities with multiple hematological tumor cell lines, with its Fc-mediated
effector functions proving essential in this context. Furthermore hz515H7 binds to primary
tumor cells from acute myeloid leukemia and multiple myeloma patients. Collectively, our
results demonstrate two major mechanisms of action, making hz515H7 unique in this regard.
Its potential as a best-in-class molecule is currently under investigation in a phase I clinical
trial.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
3
Introduction
The type-4 C-X-C-motif chemokine receptor (CXCR4) is a G-protein-coupled receptor
(GPCR) expressed predominantly in B and T cells, monocytes, natural killer and CD34+ bone
marrow progenitor cells. The only CXCR4 ligand described to date is stromal-cell-derived
factor-1 (SDF-1), also known as CXCL12 (C-X-C-motif ligand 12). SDF-1 is highly expressed
in lymph nodes, bone marrow, the liver and the lungs, and to a lesser extent in the kidneys,
brain and skin. In adults, the CXCR4/SDF-1 axis is involved in lymphocyte trafficking and in
the retention and homing of hematopoietic stem cells in the bone marrow. As with other
GPCRs, the contact between SDF-1 and the CXCR4 receptor initiates signaling via an
induced conformational change in the receptor, which is transmitted through the membrane
to promote replacement of GDP by GTP on associated G proteins (1). CXCR4 activates the
Gi/o protein family, which in turn leads to the activation of phospholipase C, the
phosphoinositide 3-kinase pathway (2), and to the regulation of gene transcription, cell
migration and cell adhesion. β-arrestin-1 and β-arrestin-2 have classically been assumed to
shut off signal transduction following receptor activation, a process termed desensitization. In
addition, β-arrestin-2 has been reported to enhance CXCR4-mediated p38 activation and cell
migration following SDF-1 stimulation (3).
Tumor tissues from at least 23 different types of human cancers of epithelial,
mesenchymal and hematopoietic origin express CXCR4 (4). Indeed, this receptor has been
found to be over-expressed in a large number of cancers including solid tumors (4–16),
leukemia (17), multiple myeloma (18) and lymphomas (19). The CXCR4/SDF-1 axis is
involved in cancer cell proliferation, in angiogenesis, and in anti-apoptotic and metastases
processes (20). CXCR4 is also expressed by cancer stem cells that have been associated
with cancer progression and treatment resistance, notably those that confer the resistance of
residual, post-chemotherapy acute myeloid leukemia (AML) blast to additional
chemotherapeutic agents, a major problem in the treatment of AML (21). These data
constitute the rationale for cancer treatments that target CXCR4. In the last few years,
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
4
clinical trials have been initiated on several CXCR4 antagonists, in combination with anti-
cancer drugs, in patients with hematologic malignancies. In particular, plerixafor, a small-
molecule CXCR4 antagonist registered by the Food and Drugs Administration in combination
with granulocyte colony-stimulating factor to enhance the mobilization of hematopoietic stem
cells to the peripheral blood for collection and subsequent autologous transplantation in
patients with lymphoma and multiple myeloma (MM), is now undergoing phase I and phase II
clinical trials for several indications including chronic lymphocytic leukemia (CLL), small
lymphocytic lymphoma, AML, MM, non-Hodgkin’s lymphoma (NHL) and Hodgkin’s disease.
Similarly, phase I clinical trials are under way for the CXCR4 human immunoglobulin IgG4
monoclonal antibody (mAb) BMS-936564, for the treatment of MM, AML, diffuse large B-cell
leukemia, CLL and follicular lymphoma. Clinical trials in phase I or II are also ongoing for the
BL-8040 (BKT140) peptide, as a treatment for chronic myeloid leukemia, MM and AML.
The above-mentioned CXCR4 antagonists can all potentially mobilize CXCR4-
expressing cancer cells, facilitating their destruction by anti-cancer drugs. Here, we describe
the generation of hz515H7, a new humanized IgG1 mAb and CXCR4 antagonist designed to
kill cancer cells via antibody- and complement-dependent cellular cytotoxicity (ADCC and
CDC), which efficiently competes for SDF-1 binding and alters the conformation of CXCR4
homodimers.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
5
Materials and Methods
Cell lines and reagents
Chinese hamster ovary (CHO)-K1 and NIH3T3 cells from the American type culture
collection (ATCC) were stably transfected to express human CXCR4. The CHO-K1 cells
were cultured in DMEM-F12 medium (Cambrex) supplemented with 5% fetal calf serum
(FCS), 1% L-glutamine and 500 µg/mL geneticin (all obtained from Gibco-Invitrogen), the
NIHT3 cells in DMEM+GlutaMAX™ medium (Gibco-Invitrogen) supplemented with 10%
FCS, 1% L-glutamine and 600 µg/mL geneticin. The HEK293 cells (from the ATCC) used for
bioluminescence resonance energy transfer (BRET) assays were cultured in
DMEM+GlutaMAX™ medium and 10% FCS (Sigma). U-937 human monocyte histiocytic
lymphoma and Ramos human B lymphoblast Burkitt lymphoma tumor cell lines were
obtained from the ATCC, and KARPAS-299 anaplastic large cell lymphoma (ALCL) human T
cell lymphoma cell line from the European collection of cell cultures. Cell lines were cultured
according to the manufacturer's instruction and were authenticated using short tandem
repeat (STR) DNA profiling (LGC Standards) on the dates indicated. U937, Ramos and
KARPAS-299 (obtained, 2009; STR, 2016). Passages were limited to 20 for experimental
procedures (<3 months) before replacing with lower passage number stocks.
Recombinant human SDF-1 was purchased from R&D Systems, [125I]-humanSDF-1
from PerkinElmer, guanosine 5’-triphosphate gamma 35S ([35S]-GTPγS) and scintillation
proximity assay (SPA) wheat germ agglutinin (WGA) beads from Amersham Biosciences.
The anti-CD20 chimeric mAb rituximab was obtained from Euromedex.
Antibodies
Anti-CXCR4 mAbs were generated by immunizing BALB/c mice (Charles River
Laboratories) subcutaneously with NIH3T3-hCXCR4 transfected cells. Supernatants of
hybridomas were evaluated for the secretion of mAbs raised against the human CXCR4
receptor by fluorescence-activated cell sorting (FACS). Selected hybridomas were cloned
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
6
using a FACS Vantage device. The best candidate mAb, 515H7, was humanized (yielding
hz515H7) using a strategy based on CDR grafting. An hz515H7 IgG1 variant with reduced
Fc-mediated effector functions was generated by mutating the leucines at positions 234 and
235 in the amino acid sequence to alanines (22). The recombinant chimeric constructs
hz515H7–mIgG2a and hz515H7–IgG4 were generated by fusing the Fc region of mouse
IgG2a and of human IgG4, to the heavy chain and light chain variable domains of hz515H7,
respectively.
Flow cytometry
The specific binding of hz515H7 to human CXCR4 was investigated by FACS.
NIH3T3 and NIH3T3-hCXCR4 transfected cells were incubated with hz515H7 or an
irrelevant hIgG1 mAb control. After incubation with a goat anti-human Alexa 488 secondary
(Invitrogen) antibody and three washing steps, propidium iodide was added to each well and
viable cells were analyzed by FACS.
To determine the expression level of CXCR4 on blasts (17 patients for AML and 8
healthy donors, Hospices Civils de Lyon, Lyon, France) and on plasmocytes (13 patients for
MM and 10 healthy donors, Hôpitaux Universitaires de Genève, Geneva, Switzerland), bone
marrow samples from patients obtained after informed consent were incubated with CD45-
V500 (clone HI30, BD560777), CD34-APC (clone 8G12, BD345804) for blasts and CD138-
PECy5 (clone BA38, CoulterA54191), CD38-V450 (clone HB7, BD646851) for plasmocytes,
as per the manufacturer’s recommendations, and with 5 µg/mL PE-labeled hz515H7 or a PE-
labeled isotype control. Two milliliters of BD (BD Biosciences) Pharm Lyse was added to lyse
red blood cells followed by washing with BD Cell Wash and resuspended in BD Cell Wash.
Samples were analyzed immediately on a BD FACSCantoII flow cytometer. Results were
expressed as a CXCR4/MFI isotype mean fluorescence intensity (MFI) ratio.
[125I]SDF-1 binding assay
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
7
A radio-ligand binding assay was performed using membranes from CXCR4
transfected CHO-K1 cells mixed with [125I]-SDF-1 (0.1 nM) and SPA-WGA beads in the
presence of indicated concentrations of hz515H7 in 96-well plates. The plates were
incubated for 1 h at room temperature (RT), centrifuged at 1,000 × g and then read on a
TopCount liquid scintillation counter (Perkin Elmer).
BRET assay of CXCR4 homodimers
HEK293 cells transfected with plasmid constructs allowing the expression either of
CXCR4 fused to R. reniformis luciferase (CXCR4-Rluc), or of both CXCR4-Rluc and CXCR4
fused to yellow fluorescent protein (CXCR4-YFP), were incubated in the presence of
antibodies (hz515H7 or a control mAb) at 20 µg/mL and 37°C prior to the addition of
coelenterazine H with or without SDF-1 (100 nM). After 5 min incubation at 37°C and 15 min
at RT, light-emission acquisition at 485 nm and 530 nm was initiated using a Mithras LB940
multimode plate reader (Berthold). BRET ratios were calculated as previously defined (23).
For each experimental point, a mean BRET ratio was calculated and expressed in milliBRET
units (mBU).
[35S]GTPγS binding assay
The [35S]GTPγS binding assay used in this study is a radioactive assay based on
scintillation proximity technology (GE Healthcare). Membranes from NIH3T3-hCXCR4
transfected cells and endogenous adaptor heterotrimeric G proteins were mixed with
unlabeled GDP, 10 nM SDF-1, SPA-WGA beads and [35S]-GTPγS in the presence of
indicated concentrations of hz515H7 mAb in 96-well plates. The plates were incubated for 1
h at RT and then read on a TopCount liquid scintillation counter (Perkin Elmer) after 10 min
RT centrifugation at 1,000 × g.
β-arrestin 2 recruitment BRET assay
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
8
The antagonist potency of hz515H7 on β-arrestin-2 recruitment following SDF-1-
induced human CXCR4 activation was evaluated using a BRET assay. CXCR4-Rluc and
CXCR4-Rluc/β-arrestin2-YFP transfected HEK293 cells were incubated for 15 min at 37°C in
the presence of different concentrations of hz515H7. The cells were incubated for a further 5
min at 37°C after adding coelenterazine H (5 µM) and SDF-1 (100 nM). Light-emission
acquisitions at 485 nm and 530 nm were then performed using a Mithras LB940 reader.
Western blot analysis
Ramos cells were incubated overnight in serum-free RPMI 1640 medium with 1% L-
glutamine, then incubated or not with hIgG1 or hz515H7 at 10 µg/mL and stimulated or not
with SDF-1 (50 ng/mL). After centrifugation, lysis buffer was added to the cells for 90 min at
4°C. After immunoprecipitation, the immunocomplexes were suspended in sample buffer,
loaded on a 4–12% SDS-PAGE and transferred to nitrocellulose blot membranes. Each blot
was saturated, incubated with appropriate (Cell Signaling) primary antibodies for 4 h at RT
and incubated with HRP-linked secondary antibodies (GE Healthcare). The blots were
subsequently assayed using the ECL detection system (Amersham Biosciences). The
membranes were then stripped, saturated and incubated with anti-GAPDH antibodies for 1 h
at RT, and finally washed and incubated with HRP-linked secondary antibodies. Optical
density maps were produced using imageJ 1.46 (24) and results expressed as an optical
density ratio after normalization with GAPDH.
Cell migration assays
High-throughput screening 96-well 8.0 µm trans-well plates (Corning Life Sciences)
were used to perform chemotaxis assays. U937 cells were suspended in RPMI medium
(BioWhittaker) and incubated for 40 min at 37°C with phosphate buffered saline, hIgG1
irrelevant antibody or hz515H7 (10 µg/mL). Then, 75 µL of cell suspension was added to the
inserts. SDF-1 was used at 800 ng/mL in a 235 µL total volume of RPMI + 2% FCS in the
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
9
bottom wells. Transwell migration was conducted for 4 h at 37°C. The cells that had migrated
into the bottom wells were quantified using ATP measurements, expressed as relative
luminescence unit (RLU).
Proliferation assay
Ramos cells were incubated in RPMI 1640 medium with 1% L-glutamine and 10%
FBS, with an IgG1 isotype control or hz515H7 at 10 µg/mL for 24 h at 37°C with or without a
cross linking antibody (a mouse anti-human IgG1 Fc antibody obtained from LSBio) added at
six-fold excess. The cells were then suspended in culture medium and incubated for three
days at 37°C. Cell viability was evaluated using ATP measurements (Cell TiterGlo,
Promega).
Cytotoxicity assays
ADCC and CDC assays on cancer cells were performed as described in refs. 25 and
26, respectively. CFSE-stained Ramos cells were spiked into blood before adding buffer,
human IgG1 isotype control, hz515H7 or rituximab at 10 µg/mL, and then incubated for 4 h at
37°C. The percentages of both viable (CFSE+ToPro3-) and dead (CFSE+ToPro3+) Ramos
cells were measured. To evaluate the effect of hz515H7 on normal blood cells, 500 µg/mL of
hz515H7, human IgG1 isotype control, or rituximab was incubated overnight at 37°C with
blood from healthy donors. The viability of B cells, T cells, monocytes and NK cells was
tracked by FACS analysis using the appropriate primary antibodies (CD19, CD3, CD14 or
CD56 from Invitrogen) and ToPro3. The results represent the percentage of viable (ToPro3−)
cells.
Binding of mAbs to the mouse Fc gamma receptor IV
Real-time surface plasmon resonance interaction assays were carried out using a
Biacore X device and reagents supplied by Biacore (GE Healthcare). To study the binding of
mAbs on the mouse FcγRIV extracellular domain (ECD, Sino Biological Inc.), 1033.5 RU of
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
10
the m-FcγRIV ECD was chemically grafted on the FC2 of a CM4 sensor chip (GE
Healthcare) using the amine coupling kit. The first flow cell (FC1) served as the reference
surface to subtract the non-specific interactions. Kinetic experiments were carried out at
25°C a flow rate of 30 µL/min. An injection of HBS-EP running buffer analyte was used as a
double reference. The binding of mAbs to mouse FcγRIV was evaluated using BIAevaluation
3.1 (GE Healthcare).
Mouse models
Ten million Ramos cells were engrafted subcutaneously to severe combined
immunodeficiency (SCID) mice (Charles River Laboratories). After eight days, the mice were
randomized into groups with mean tumor sizes of 100 mm3. The mice were treated weekly by
intraperitoneal injections with appropriate doses (0.15–20 mg/kg) of hz515H7 with a loading
dose. Five million KARPAS-299 cells were engrafted subcutaneously to non-obese diabetic
(NOD) SCID mice (Charles River Laboratories). Six days after implantation, the mice were
randomized into two groups with mean tumor sizes of 130 mm3. The mice were treated by
intraperitoneal injections of hz515H7 at 40 mg/kg (loading dose) and then weekly at 20
mg/kg. In both models, a histidine buffer group was introduced as control. Tumor volumes
were measured twice a week with an electronic caliper and calculated with the formula: π/6 ×
length × width × height.
Ten million U-937 cells were engrafted intraperitoneally on seven-week-old
NOD/SCID or NOD/SCID gamma (NSG) female mice. The antibodies were administered
subcutaneously, starting two days after cell engraftment, with a loading dose of 20 mg/kg,
followed by weekly injections of a 10 mg/kg dose. The efficacy of the treatment was
measured by monitoring the mouse survival rate daily, and by comparing the treated groups
with the control untreated group (injected with histidine buffer).
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
11
Results
hz515H7 recognizes human CXCR4, antagonizes SDF-1 binding and induces a
conformational change in CXCR4 homodimers
The binding specificity of hz515H7 on both NIH3T3 wild-type cells and NIH3T3-
hCXCR4 transfected cells was evaluated by FACS analysis. Neither hz515H7 nor hIgG1
bound wild-type NIH3T3 parent cells (see insert in Figure 1A). In contrast, concentration-
dependent binding of hz515H7 (but not of hIgG1) was observed on the surface of NIH3T3-
hCXCR4 transfected cells (Fig. 1A). The Kd value of the mAb measured on NIH3T3-hCXCR4
transfected cells was 0.290 ± 0.009 nM. Furthermore, to confirm CXCR4 specificity,
hz515H7-1 was evaluated for its ability to recognize human cell lines by FACS analysis. This
profile of cells recognition was compared with the one obtained by qPCR quantifying mRNA
encoding for hCXCR4. hz515H7-1 was able to recognize cells for which mRNA encoding for
hCXCR4 was quantified but not cells for which mRNA encoding for hCXCR4 was not
detected (data not shown).
In order to evaluate the ability of hz515H7 to compete with SDF-1 for CXCR4 binding,
saturation-binding experiments were conducted using radio-labeled [125I]-SDF-1 and
membranes of CHO-K1 cells stably expressing human CXCR4. Hz515H7 inhibited [125I]SDF-
1 binding concentration-dependently with a half-maximal inhibitory concentration (IC50) of
approximately 1.5 nM (Fig. 1B). As reported previously (27,28), we used a BRET assay to
monitor for ligand-induced conformational changes in CXCR4 homodimers (Fig. 1C). The
addition of SDF-1 (100 nM) induced an increase in the BRET signal, reflecting a
conformational change. In contrast, the binding of hz515H7 (20 µg/mL) to CXCR4
homodimers led to a decrease in the BRET signal both in the absence and presence of SDF-
1. These results suggest that the binding of hz515H7 to a CXCR4 homodimer induces a
conformational change in the receptor that prevents any further changes induced by SDF-1.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
12
hz515H7 recognizes CXCR4 on primary blasts and plasmocytes from AML and MM
patients
To evaluate CXCR4 expression on primary leukemic blasts and malignant
plasmocytes, we stained CXCR4 in fresh bone marrow samples from AML and MM patients
with PE-labeled hz515H7 or PE-isotype control (18 healthy donors). For 6 out of 17 (~35%)
samples from AML patients and for 9 out of 13 (~69%) from MM patients, the surface
expression levels of CXCR4 of blast cells (CD45Dim, CD34+) and of plasmocytes (CD38+,
CD138+) were higher than those of normal CD34+ cells in healthy BM samples (Table1).
hz515H7 inhibits both G-protein dependent and β-arrestin-2 dependent SDF-1-
mediated signaling pathways
Following SDF-1 binding, the conformation adopted by CXCR4 leads to the activation
of heterotrimeric G proteins associated with an exchange between bound GDP and GTP
from the Gα subunit (29). In a first assay, the incorporation of radio-labeled [35S]GTPγS was
monitored as a proxy for the activation level of CXCR4. The incubation of hz515H7 with
SDF-1 (10 nM)-activated membranes of NIH3T3-CXCR4 inhibited [35S]GTPγS binding by up
to 89% in a concentration dependent manner (IC50 3.5 nM) (Fig. 2A).
The binding of SDF-1 to CXCR4 induces its internalization through the recruitment of
β-arrestin and clathrin-mediated endocytosis (30). The BRET assay in Fig. 2B demonstrates
that hz515H7 inhibits β-arrestin-2 recruitment in response to SDF-1 stimulation with an IC50
of 3 nM. Furthermore, Fig. 2C clearly shows that within 5 min, hz515H7 dramatically reduces
the SDF-1-induced phosphorylation of Akt, Erk1/2, p38 and GSK3β. This inhibition is
maintained for at least 60 min (Fig. 2D). Altogether, our results indicate that hz515H7 inhibits
both G-protein- and β-arrestin-2-dependent SDF-1-mediated intracellular signaling pathways.
Finally, Fig. 3 demonstrates that hz515H7 inhibits SDF-1-induced U937 cell migration and
proliferation in the presence of a crosslinking antibody by 80.0 ± 12.0 % (Fig. 3A) and ~50%
(Fig. 3B), respectively.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
13
hz515H7 induces both ADCC and CDC of tumor cells but has no impact on normal
blood cells in vitro
Antibody- and complement-dependent cytotoxicity are important mechanisms in mAb
cancer therapy (31). The ability of hz515H7 to trigger ADCC was assessed on Ramos cells
using purified NK cells from healthy donors. As shown in Fig. 3C, 10 µg/mL of the hz515H7
IgG1 mAb induced 50 ± 10% cell cytotoxicity whereas the IgG1 control mAb and the
hz515H7 IgG4 mAb which binds tumor cells in a manner similar to that of the corresponding
IgG1 antibody, did not lead to ADCC.
Next, we investigated CDC at different culture times in Ramos cells expressing
different levels of CXCR4. Hz515H7 induced concentration dependent cell lysis (Fig. 3D).
Interestingly, there is a strong correlation between the concentration response curves of
CDC and of hz515H7 binding to Ramos cells. The EC50s for both assays are similar (1.4
×10−10 M and 1.6 × 10−10 M, respectively). The absence of cell lysis in the presence of heated
serum (Fig. 3E) confirms the involvement of the complement in the CDC assay. As expected,
the hz515H7 IgG4 mAb did not trigger any CDC on Ramos cells (Fig. 3F).
Regarding the elimination of tumor cells in blood, Fig. 4A shows that over 90% of the
Ramos cells expressing both CXCR4 and CD20 are killed in the presence of either hz515H7
or rituximab, but not in the presence of a control isotype. In addition, Fig. 4B reveals that
whereas rituximab (a positive control) causes B cell depletion, hz515H7 has no major impact
on normal B-cells, T-cells, monocytes or NK cells. These results indicate that in vitro,
hz515H7 triggers Ramos cancer cell lysis in whole blood while preserving normal cells.
Efficacy of hz515H7 in xenograft mice tumor models and the role of effector functions
in the activity of this mAb
The activity of hz515H7 against tumor growth was evaluated in Ramos human B
Burkitt lymphoma and KARPAS-299 ALCL xenograft models. In the Ramos model (Fig. 5A)
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
14
hz515H7 inhibited tumor growth in a dose-dependent manner, by as much as 98% at 20
mg/kg at day 23. Tumor growth was also significantly inhibited (by as much as 68%) in the
KARPAS-299 model (Fig. 5B). It has to be noted that, as hz515H7 does not recognize the
murine CXCR4, it does not allow in these mice models, the measurement of the activity of
mAb on endothelial cells in the process of angiogenesis and the potential toxicity on the
different normal tissues expressing CXCR4.
Finally, the importance of effector functions in hz515H7’s mechanism of action was
investigated with a U937 AML xenograft tumor model in NOD/SCID mice. The effects of
hz515H7 IgG1 were compared on the one hand with chimeric mouse-human mIgG2a
hz515H7, designed to have optimal effector functions in mice, and on the other with an
hz515H7 IgG1 Ala/Ala mutant designed to have reduced effector function (32). Figure 6A
clearly shows that mIgG2a hz515H7 and hz515H7 IgG1 bind very efficiently to mouse
FcγRIV while the Ala/Ala mutant does not. The survival rates of mice treated with the two
former constructs are much higher than those of mice treated with the Ala/Ala mutant or in
the control group (Fig. 6B). Furthermore as displayed in Fig. 6C, hz515H7 has virtually no
anti-tumor activity in NSG mice lacking the IL2 receptor gamma chain. These results
therefore demonstrate that the effector functions of hz515H7 are essential for its antitumor
activity.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
15
Discussion
Despite huge progress in the treatment of cancer patients, residual disease still leads
to cancer relapse and mortality. CXCR4 is known to contribute to microenvironment-
mediated chemoresistance, an important barrier to the eradication of residual disease that
has yet to be overcome, notably when bone marrow niches offer protection to cancer cells
(33,34). The value of targeting CXCR4 with IgG4 mAbs (35), peptides (36) and small
molecules (37) has already been demonstrated in pharmacological models. Indeed, these
agents mobilize cancer cells thereby facilitating their elimination by chemotherapeutic
agents. These CXCR4 antagonists are now under investigation in clinical trials. Nonetheless,
it may be possible to further increase CXCR4 antagonist efficacy. For example, Kularatne et
al. (38) recently described an antibody-drug conjugate strategy to selectively deliver the
cytotoxic agent to cancer cells using a CXCR4 mAb coupled to monomethyl auristatin F,
which allowed the selective elimination of CXCR4+ metastatic cancer cells both in vitro and
in vivo. Importantly, this approach was also shown to spare CXCR4+ hematopoietic cells in
vivo. The new humanized IgG1 mAb reported here, hz515H7, binds the human chemokine
receptor CXCR4, competes for SDF-1 binding and induces a conformational change in
CXCR4 homodimers. It inhibits both CXCR4 receptor-mediated G-protein activation and β-
arrestin-2 recruitment following CXCR4 activation. The binding of hz515H7 to CXCR4 inhibits
SDF-1-induced signaling pathways and the migration and proliferation of cancer cells. These
properties are similar to those of other CXCR4/SDF1 antagonists currently under clinical trial.
In addition however, this study demonstrates that hz515H7 induces cell lysis in cancer cells
in vitro, via ADCC and CDC. Moreover, our findings for multiple hematological tumor
xenograft models suggest that these in vitro properties translate into a strong anti-tumor
activity in vivo. We also evaluated the in vivo anti-tumor activity of three different isotypes of
hz515H7: the IgG1 wild-type, an Ala/Ala IgG1 mutant with reduced effector functions, and a
chimeric mouse-human construct, mIgG2a hz515H7, designed to have optimal effector
functions in mice. The dramatic improvement in survival rates observed here in a U937
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
16
survival model for mice treated with hz515H7 IgG1 and mIgG2a hz515H7 demonstrates the
importance of effector functions for the anti-tumor activity of hz515H7, the Ala/Ala mutant
losing most of its in vivo activity, and hz515H7 also proving significantly less efficient in NSG
mice lacking NK cell activity.
Several other studies have demonstrated the importance of CDC and ADCC for the
therapeutic efficacy of mAbs, particularly for rituximab, a CD20 mAb largely used in the
treatment of NHL and CLL (39, 40). Daratumumab, a CD38 mAb, kills MM tumor cells by
inducing CDC and ADCC (41) and is a therapeutic mAb with high potential for the treatment
of MM patients. Finally, elotuzumab, a CS1 mAb recently approved by the FDA for the
treatment of MM patients, has been shown to inhibit the binding of MM cells to bone marrow
stromal cells and to induce ADCC in MM cells (42).
Our results also show that the CDC induced by hz515H7 is correlated with the
expression of CXCR4 on the cell membrane of Ramos cells (Fig. 2). Similar to previous
reports (43,18), we found that CXCR4 is over-expressed in cancer cells, in respectively 35%
and ~69% of samples from AML and MM patients, with expression levels of CXCR4 on blast
cells and of plasmocytes higher than those of CD34+ cells in healthy BM samples,
suggesting that hz515H7 should preferentially kill the neoplastic cells while preserving
normal hematopoietic cells. Indeed, our in vitro results show that hz515H7 does not
decrease the viability of B-cells, monocytes, T-cells or NK cells, while rituximab, used as a
positive control for ADCC (44) and CDC (40), leads to CD20+ B cell depletion as previously
described (44, 45).
The two major mechanisms of action demonstrated here for hz515H7, namely
interference with the CXCR4/SDF-1 axis and the triggering of effector functions (ADCC and
CDC), make it unique among mAbs. A phase I clinical trial is under way on this potentially
best-in-class molecule for the treatment of hematologic malignancies. Furthermore, as
CXCR4 is expressed in solid tumors, hz515H7 is also being investigated for its anti-tumor
activity in solid xenograft models.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
17
Acknowledgments
The authors thank C. Tardy, A. Blaecke, M. Fournier, S Bernois, C. Bertaux, K. Leveque, V.
Bonnet, L. Revy, C. Daviet, L. Troncy. P. Aspinion, M. Excoffier, MC. Janin-Bussat for their
technical assistance and C. Catry and A. Dorison for their clerical support.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
18
References
1. Li JH, Hamdan FF, Kim SK, Jacobson KA, Zhang X, Han SJ et al. Ligand-specific
changes in M3 muscarinic acetylcholine receptor structure detected by a disulfide
scanning strategy. Biochemistry. 2008;47:2776-88.
2. Busillo JM, Benovic JL. Regulation of CXCR4 signaling. Biochim Biophys Acta
2007;1768:952–63.
3. Sun Y, Cheng Z, Ma L, Pei G. β-arrestin2 is critically involved in CXCR4-mediated
chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J Biol
Chem 2002;277:49212–19.
4. Balkwill F. The significance of cancer cell expression of the chemokine receptor CXCR4.
Semin Cancer Biol 2004;14:171–9.
5. Ottaiano A, Franco R, Aiello Talamanca A, Liguori G, Tatangelo F, Delrio P et al.
Overexpression of both CXC chemokine receptor 4 and vascular endothelial growth
factor proteins predicts early distant relapse in stage II-III colorectal cancer patients. Clin
Cancer Res 2006;12:2795–803.
6. Kato M, Kitayama J, Kazama S, Nagawa H. Expression pattern of CXC chemokine
receptor-4 is correlated with lymph node metastasis in human invasive ductal carcinoma.
Breast Cancer Res 2003;5:144–50.
7. Sun YX, Wang J, Shelburne CE, Lopatin DE, Chinnaiyan AM, Rubin MA, et al.
Expression of CXCR4 and CXCL12 (SDF-1) in human prostate cancers (PCa) in vivo. J
Cell Biochem 2003;89:462–73.
8. Phillips RJ, Burdick MD, Lutz M, Belperio JA, Keane MP, Strieter RM. The stromal
derived factor-1/CXCL12-CXC chemokine receptor 4 biological axis in non-small cell lung
cancer metastases. Am J Respir Crit Care Med 2003;167:1676–86.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
19
9. Scotton CJ, Wilson JL, Scott K, Stamp G, Wilbanks GD, Fricker S, et al. Multiple actions
of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res
2002;62:5930–8.
10. Koshiba T, Hosotani R, Miyamoto Y, Ida J, Tsuji S, Nakajima S et al. Expression of
stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a
possible role for tumor progression. Clin Cancer Res 2000;6:3530–5.
11. Wang L, Wang L, Yang B, Yang Q, Qiao S, Wang Y et al. Strong expression of
chemokine receptor CXCR4 by renal cell carcinoma cells correlates with metastasis Clin
Exp Metastasis 2009;26:1049–54.
12. Barbero S, Bajetto A, Bonavia R, Porcile C, Piccioli P, Pirani P et al. Expression of
chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 in human brain
tumors and their involvement in glial proliferation in vitro. Ann NY Acad Sci 2002;973:60–
9.
13. Bian XW, Yang SX, Chen JH, Ping YF, Zhou XD, Wang QL, et al. Preferential expression
of chemokine receptor CXCR4 by highly malignant human gliomas and its association
with poor patient survival. Neurosurgery 2007;61:570–9.
14. De Falco V, Guarino V, Avilla E, Castellone MD, Salerno P, Salvatore G et al. Biological
role and potential therapeutic targeting of the chemokine receptor CXCR4 in
undifferentiated thyroid cancer. Cancer Res 2007;67:11821–9.
15. Scala S, Giuliano P, Ascierto PA, Ieranò C, Franco R, Napolitano M et al. Human
melanoma metastases express functional CXCR4. Clin Cancer Res 2006;12:2427–33.
16. Meng X, Wuyi L, Yuhong X, Xinming C. Expression of CXCR4 in oral squamous cell
carcinoma: correlations with clinicopathology and pivotal role of proliferation. J Oral
Pathol Med 2010;39:63–8.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
20
17. Burger JA, Bürkle A. The CXCR4 chemokine receptor in acute and chronic leukaemia: a
marrow homing receptor and potential therapeutic target. Br J Haematol 2007;137:288–
96.
18. Trentin L, Miorin M, Facco M, Baesso I, Carraro S, Cabrelle A et al. Multiple myeloma
plasma cells show different chemokine receptor profiles at sites of disease activity. Br J
Haematol 2007;138:594–602.
19. Trentin L, Cabrelle A, Facco M, Carollo D, Miorin M, Tosoni A et al. Homeostatic
chemokines drive migration of malignant B cells in patients with non-Hodgkin
lymphomas. Blood 2004;104:502–8.
20. Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res
2010;16:2927–31.
21. Calandra G, Bridger G, Fricker S. CXCR4 in Clinical Hematology Curr Top Microbiol
Immunol 2010;341:173–91.
22. Hezareh M, Hessell AJ, Jensen RC, Van De Winkel JGJ, Parren PWHI. Effector function
activities of a panel of mutants of a broadly neutralizing antibody against human
immunodeficiency virus type 1. J Virol 2001; 75: 12161–8.
23. Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M et al. Detection of beta 2-
adrenergic receptor dimerization in living cells using bioluminescence resonance energy
transfer (BRET). Proc Natl Acad Sci USA 2000;97:3684–89.
24. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image
analysis. Nat Methods 2012;9:671–5.
25. Broussas M, Broyer L, Goetsch L. Evaluation of antibody-dependent cell cytotoxicity
using lactate dehydrogenase (LDH) measurement. Methods Mol Biol 2013; 988:305–17.
26. Broyer L, Goetsch L, Broussas M. Evaluation of complement-dependent cytotoxicity
using ATP measurement and C1q/C4b binding. Methods Mol Biol 2013;988:319–29.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
21
27. Toth PT, Ren D, Miller RJ. Regulation of CXCR4 receptor dimerization by the chemokine
SDF-1alpha and the HIV-1 coat protein gp120: a fluorescence resonance energy transfer
(FRET) study. J Pharmacol Exp Ther 2004;310:8–17.
28. Percherancier Y, Berchiche YA, Slight I, Volkmer-Engert R, Tamamura H, Fujii N,
Bouvier M, Heveker N. Bioluminescence resonance energy transfer reveals ligand-
induced conformational changes in CXCR4 homo- and heterodimers. J Biol Chem 2005;
280:9895–903.
29. Radhika V, Dhanasekaran N. Transforming G proteins. Oncogene. 2001;20:1607–14.
30. McCormick PJ, Segarra M, Gasperini P, Gulino AV, Tosato G. Impaired recruitment of
Grk6 and beta-Arrestin 2 causes delayed internalization and desensitization of a WHIM
syndrome-associated CXCR4 mutant receptor. PLoS One. 2009;4:e8102.
31. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer
2012;12:278–87.
32. Xu D, Alegre ML, Varga SS, Rothermel AL, Collins AM, Pulito VL et al. In vitro
characterization of five humanized OKT3 effector function variant antibodies. Cell
Immunol 2000;200:16–26.
33. Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk between tumor cells and
their microenvironment. Blood 2006;107:1761–7.
34. Zeng Z, Shi YX, Samudio IJ, Wang RY, Ling X, Frolova O et al. Targeting the leukemia
microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and
chemotherapy in AML. Blood 2009;113:6215–24.
35. Kuhne RM, Mulvey T, Belanger B, Chen S, Pan C, Chong et al. BMS-936564/MDX-
1338: a fully human anti-CXCR4 antibody induces apoptosis in vitro and shows antitumor
activity in vivo in hematologic malignancies. Clin Cancer Res 2013;19:357–66.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
22
36. Beider K, Begin M, Abraham M, Wald H, Weiss ID, Wald O et al. CXCR4 antagonist 4F-
benzoyl-TN14003 inhibits leukemia and multiple myeloma tumor growth. Exp Hematol
2011;39:282–92.
37. Azab AK, Runnels JM, Pitsillides C, Moreau AS, Azab F, Leleu X et al. CXCR4 inhibitor
AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow
microenvironment and enhances their sensitivity to therapy. Blood 2009;113:4341–51.
38. Kularatne SA, Deshmukh V, Ma J, Tardif V, Lim RKV, Pugh HM et al. A CXCR4-targeted
site-specific antibody-drug conjugate. Angew Chem Int Ed 2014;53:11863–7.
39. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo
cytoxicity against tumor targets. Nat Med 2000;6:443–6.
40. Di Gaetano N, Cittera E, Nota R, Vecchi A, Grieco V, Scanziani E et al. Complement
activation determines the therapeutic activity of rituximab in vivo. J Immunol
2003;171:1581–7.
41. De Weers M, Tai YT, van der Veer MS, Bakker JM, Vink T, Jacobs CDH et al.
Daratumumab, a novel therapeutic human CD38 monoclonal antibody, induces killing of
multiple myeloma and other hematological tumors. J Immunol 2011;186:1840–8.
42. Tai YT, Dillon M, Song W, Leiba M, Li XF, Burger P. Anti-CS1 humanized monoclonal
antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent
cellular cytotoxicity in the bone marrow milieu. Blood 2008; 112: 1329–37.
43. Spoo AC, Lübbert M, Wierda WG, Burger JA. CXCR4 is a prognostic marker in acute
myelogenous leukemia Blood 2007;109:786–91.
44. Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R et al. Depletion of B
cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood
1994;83:435–45.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
23
45. Vugmeyster Y, Howell K, Bakshl A, Flores C, Canova-Davis E. Effect of anti-CD20
monoclonal antibody, Rituxan, on cynomolgus monkey and human B cells in a whole
blood matrix. Cytometry A 2003;52:101–9.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
24
Table 1: CXCR4 expression on blasts (AML patients) and on plasmocytes (MM patients)
AML patients blast cells MM patients plasma cells Patient
#MFI
isotype MFI CXCR4 MFI Ratio
(a) Patient #
MFI isotype MFI
CXCR4 MFI Ratio (b)
1 61 4334 71 1 49 72 2
2 106 360 3 2 47 78 2
3 108 334 3 3 32 32 1
4 119 478 4 4 3 33 11
5 778 1559 2 5 3 64 20
6 97 817 8 6 1 7 7
7 83 3815 46 7 2 29 15
8 86 3291 38 8 2 3 2
9 123 2410 20 9 6 49 8
10 196 4407 22 10 8 44 6
11 74 927 13 11 2 16 8
12 126 1900 15 12 2 26 13
13 48 435 9 13 3 39 13
14 93 239 3
15 65 1094 17
16 116 1428 12
17 165 4419 27
(a) MFI ratio is 10 ± 6 for blast cells from healthy donors (b) MFI ratio is 2.6 ± 0.9 for CD34
+ cells from healthy donors
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
25
Figure legends
Figure 1. Hz515H7 recognizes human CXCR4, antagonizes SDF-1 binding and induces a
conformational change in CXCR4 homodimers. (A) FACS analysis of viable NIH3T3 and
NIH3T3-hCXCR4 transfected cells incubated with hz515H7 or an irrelevant hIgG1 mAb. (B)
Membranes from CXCR4 transfected CHO-K1 cells in the presence of hz515H7, using SDF-
1 as a competitor ligand. The data points represent the means of three independent
experiments, two sets of which were performed for each point. (C) BRET analysis (% of the
basal BRET signal), of HEK293 cells expressing CXCR4-Rluc or both CXCR4-Rluc and
CXCR4 fused to YFP, incubated in the presence of antibodies (hz515H7 or a control mAb)
with or without SDF-1 (100 nM). The data shown are means of at least five repeat
measurements, and the bars represent the associated standard errors.
Figure 2. Hz515H7 inhibits the G-protein-dependent and β-arrestin-2 signaling pathways
induced by SDF-1. (A) Inhibition of SDF-1-induced [35S]GTPγS binding to membranes of
NIH3T3-hCXCR4 transfected cells as a function of hz515H7 concentration. Each data point
is the mean of three experiments, and measurements at each concentration were performed
in duplicate. (B) BRET analysis, following the addition of coelenterazine H (5 µM), of CXCR4-
Rluc and CXCR4-Rluc/β-arrestin2-YFP expressing HEK293 cells incubated in the presence
of hz515H7 with or without SDF1 (100 nM). The data are means of seven experimental
values and the bars represent the associated standard errors. (C, D) Immunoblot analysis of
Ramos cells incubated or not with hIgG1 or hz515H7 at 10 µg/mL and stimulated or not with
SDF-1 (50 ng/mL) for the time periods indicated in part (C). (D) Optical density (OD) ratios,
using GAPDH and the corresponding protein to normalize, obtained from OD maps produced
using imageJ software.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
26
Figure 3. Hz515H7 inhibits U937 cancer cell migration and Ramos cell proliferation. (A) U-
937 cells migration after incubation in phosphate buffered saline (PBS), 10 µg/mL hIgG1
isotype control or hz515H7, in the presence or absence of SDF-1 (800 ng/mL). The data
shown are the mean and standard deviations of three independent measurements, each
performed in triplicate. (B) Percentage inhibition of Ramos cells proliferation after incubation
for 72 h at 37°C either in medium (RPMI 1640 with 1% L-glutamine and 10%FBS) by itself, or
with an IgG1 isotype control or hz515H7 at 10 µg/mL, with or without cross-linking antibodies
(viz. anti-human IgG1 Fc antibodies). The data shown are the mean of two independent
experiments, each performed in duplicate, and the associated standard errors. (C–F)
Hz515H7-induced ADCC and CDC in tumor cells. (C) Mean cytotoxicity percentage (±
standard deviations) of Ramos cells pre-incubated with 10 µg/mL hIgG1 isotype control,
hz515H7 IgG1 or hz515H7 IgG4. Purified NK cells from healthy donors were used as effector
cells. (D) FACS analysis of CXCR4 expression in Ramos cells pre-incubated with increasing
concentrations of hz515H7 IgG1 before human sera were added. (E) Mean cytotoxicity
percentage (± standard deviations) of Ramos cells pre-incubated with 10 µg/mL hIgG1
isotype control or hz515H7 IgG1, in heated or non-heated human or baby rabbit sera (10% of
the final concentration), to evaluate CDC. (F) Mean cytotoxicity percentage (± standard
deviations) of Ramos cells pre-incubated with buffer, 10 µg/mL hIgG1 isotype control,
hz515H7 IgG1 or hz515H7 IgG4 before adding human serum.
Figure 4. Hz515H7 does not affect normal blood cells in vitro. (A, B) Viability (as measured
by FACS) (A) of CFSE-stained Ramos cells spiked into blood before adding buffer, a human
IgG1 isotype control mAb, hz515H7 or rituximab at 10µg/mL, and then incubated for 4 h at
37°C; and (B) of B cells, T cells, monocytes and NK cells in blood from healthy donors (n=6)
incubated overnight at 37°C in the presence of a human IgG1 isotype control mAb, hz515H7,
or rituximab at 500 µg/mL
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
27
Figure 5. Efficacy of hz515H7 in xenograft mice tumor models. (A, B) Evolution of tumor
volumes in (A) seven-week-old female SCID mice engrafted subcutaneously with 107 Ramos
cells, and treated intraperitoneally (n=6) with appropriate doses of hz515H7 or a histidine
buffer after eight days and then once a week for five weeks; and (B) seven-week-old
NOD/SCID mice engrafted subcutaneously with 5 × 106 KARPAS-299 cells and treated
intraperitoneally (n=6) with a histidine buffer or hz515H7 at 40 mg/kg after six days and then
once a week at 20 mg/kg. A Mann-Whitney statistical test was performed after each
measurement. The data from one experiment representative of at least two are shown.
Figure 6. The role of effector functions in the in vivo activity of hz515H7. (A) Binding, as a
function of time, of chimeric mouse-human mIgG2a hz515H7, hz515H7 hIgG1 and hz515H7
hIgG1 Ala/Ala mutant mAbs to the mouse FcγRIV. (B, C) Survival rates of (B) seven-week-
old NOD/SCID and (C) NSG female mice engrafted intraperitoneally with 107 U937 cells and
injected subcutaneously with histidine buffer, mIgG2a hz515H7, hz515H7 hIgG1, or hz515H7
hIgG1 Ala/Ala mutant at 20 mg/kg (loading dose), then weekly at 10 mg/kg. At the end of the
experiment, the data were analyzed using a survival log rank test. The data from one
experiment representative of two are shown.
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
Figure 1 on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
Figure 2 on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
Figure 3 on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
Figure 4
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
Figure 5
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
Time (s)
Resp
. D
iff.
RU
-25
25
75
125
175
-30 0 30 60 90 120 150 180 210 240 270 300
hz515H7 hIgG1
hz515H7 mIgG2a
hz515H7 hIgG1A/A
HBS-EP
A
Figure 6 on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041
Published OnlineFirst June 13, 2016.Mol Cancer Ther Mattieu Broussas, Nicolas Boute, Barbara Akla, et al. and mobilizes effector cellsA new anti-CXCR4 antibody that blocks the CXCR4/SDF-1 axis
Updated version
10.1158/1535-7163.MCT-16-0041doi:
Access the most recent version of this article at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://mct.aacrjournals.org/content/early/2016/06/11/1535-7163.MCT-16-0041To request permission to re-use all or part of this article, use this link
on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041