A new anti-CXCR4 antibody that blocks the CXCR4/SDF-1 axis and … · 2016. 6. 11. · 1 Title A new anti-CXCR4 antibody that blocks the CXCR4/SDF-1 axis and mobilizes effector cells

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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

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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.




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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,




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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.




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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




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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




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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




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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




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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




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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).




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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.




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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.




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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)




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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.




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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




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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.




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.




18

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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




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.




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




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.




Figure 1 on July 21, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from









Figure 4




Figure 5




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




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

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A new anti-CXCR4 antibody that blocks the CXCR4/SDF-1 axis and … · 2016. 6. 11. · 1 Title A new anti-CXCR4 antibody that blocks the CXCR4/SDF-1 axis and mobilizes effector cells