Download SAR650984 binding and activity on human blood cells

Supplementary Data
Supplementary Methods
SAR650984 binding and activity on human blood cells
All human blood samples were obtained after written informed consent under an
institutional review board approved protocol. Fresh buffy coats or whole blood samples
from healthy donors were obtained from Research Blood Components (Brighton, MA).
Peripheral blood mononuclear cells (PBMCs) were prepared by standard Ficoll-Paque
centrifugation. For CD38 and CD20 expression analysis, 2 x 105 cells were stained for
indicated blood cell markers (Miltenyi) together with 16 μg/mL of anti-CD20-PE (BD
Biosciences) or 2.5 μg/mL of anti-CD38-PE for 1 hour on ice in PBS with 0.5% BSA and
20% human AB serum (Sigma-Aldrich). Samples were analyzed in conjunction with
QuantiBRITE™ beads as described for Expression analysis of cell lines.
For binding affinity, 2 x 105 PBMCs were stained with anti-CD14 or anti-CD3 antibodies
together with AF488-labeled SAR650984 or AF488-labeled huIgG1 isotype control
antibody at concentrations ranging from 1.4 x 10-8 M to 7.1 x 10-13 M for 1 hour on ice in
PBS with 0.5% BSA and 20% human AB serum (Sigma-Aldrich). Samples were washed
twice with staining buffer, fixed in 500 μL of 1% formaldehyde in 1x PBS and analyzed
by flow cytometry.
For in vitro cell depletion, 5 x 105 PBMCs were incubated with 10 µg/mL of SAR650984,
rituximab or alemtuzumab for 1 hour at 37°C. Samples were co-stained for indicated
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blood cell markers and analyzed by flow cytometry with CountBright Absolute Counting
Beads (Invitrogen) to standardize cell counts. Depletion was determined as: Percent
Depletion = 100 x (1 – cell-to-bead ratio of treated sample/ cell-to-bead ratio of
controls).
Enzymatic activity assays
An expression plasmid was constructed that allows expression of amino acids 44-300 of
human CD38 fused to the Fc portion of murine IgG2a. The CD38-Fc fusion protein was
expressed by transient transfection in HEK-293T cells using the CalPhos Mammalian
Transfection Kit according to the manufacturer’s instructions (BD Biosciences). The
resulting supernatant was purified by standard protein-A chromatography (GE
Healthcare). Protein concentration was determined by A280 using the predicted molar
extinction coefficient of 85920. CD38 catalyzes the metabolism of nicotinamide adenine
dinucleotide (NAD) phosphate and cyclic adenosine diphosphate ribose (cADPR). Since
cADPR hydrolysis is generally very fast, an alternative enzymatic assay with a slower
kinetics has been developed with nicotinamide guanine dinucleotide (NGD)+ as a
substrate instead of NAD+. NGD+ is converted to cyclic GDP-ribose (cGDPR) and
nicotinamide followed by cGDPR hydrolysis to GDPR. cGDPR hydrolysis is slow in
vitro, leading to cGDPR accumulation. The enzymatic activity of a NADase can be
monitored by detection of the fluorescent product, cGDPR. Anti-CD38 antibodies were
pre-incubated at a final antibody concentration of 200, 20, 2, and 0.2 nM (equal to 0, 3,
0.3 and 0.03 μg/ml) with 5 nM recombinant CD38 protein for 15 minutes at room
temperature (RT) in a black 96-well plate in reaction buffer (20mM Tris-HCl, pH 7.0).
NGD substrate solution (NGD sodium salt, Sigma Aldrich) in reaction buffer was added
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to reach a final concentration of 80 μM. The reaction was incubated at room
temperature in the dark and production of cGDPR was monitored by measuring the
fluorescence signal at an excitation wavelength of 300 nm (EX300) and an emission
wavelength of 410 nm (EM400) over time. Three independent assays were performed
with duplicate wells for each treatment. Readings from wells without CD38-Fc were
used as background values and subtracted from all the sample readings. Backgroundcorrected fluorescence readings for EX300/EM410 were plotted in Relative
Fluorescence Units (RFU) for each antibody concentration versus the reaction time.
Percent cGDPR production was calculated relative to control samples in the absence of
antibody (no Ab) by the following formula: %cGDPR production = (RFU of sample/
mean RFU of no Ab control) *100.
Detection of cleaved caspase 7
Induction of cleaved caspase 7 was evaluated in SU-DHL-8 tumors excised 14 or 24
hours after a single administration of 40 mg/kg SAR650984. Treated and untreated
tumor samples were lysed in buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% Triton, 1 mM NaF, 20 mM Na4P2O7, 1 mM activated Na3VO4,
10% glycerol supplemented with protease inhibitors). Samples were incubated for 2
hours at 4°C and clarified by centrifugation at 16,000 x g for 15 minutes at 4°C.
Supernatants were collected and analyzed by Immunoblot using rabbit anti-cleaved
caspase 7 (CST#9491) or rabbit anti-β-actin (CST#4970, Cell Signaling Technologies)
as a loading control. Signal on blots was quantitated using Multi Gauge software
(Fujifilm) in arbitrary signal intensity units (SIU). The percentage of cleaved caspase 7
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was calculated at for each time point as follows: % cleaved caspase 7 = 100x (SIU of
SAR650984 treated tumors - SIU of control tumors)/ SIU of control tumors.
Preparation of the SAR650984-Fab fragment
In order to allow transient expression of the Fab fragment of the humanized anti-CD38
antibody SAR650984 in HEK293-FS™ cells, two expression vectors encoding
respectively the light chain and a CH2 and CH3 Fc deleted form of heavy chain were
generated. The DNA sequence encoding the light chain of SAR650984 was cloned, in
frame and downstream of a signal peptide (MGWSCIILFVATATGVHS from the mouse
Igh-VJ558 gene), into a mammalian expression vector derived from pCEP4 (Life
Technologies) resulting in the light chain expression plasmid pBH3045.
The DNA sequence encoding the VH-CH1 region of the heavy chain (or Fc deleted
heavy chain) of SAR650984 was PCR amplified and a Hisx6 tag was introduced in
frame to allow expression of the SAR650984HC-Hisx6 fusion protein. Partial NheI and
HindIII sites were added at 5’ ends of forward and reverse primers respectively, which
allow cloning PCR fragments directly into a mammalian expression vector digested with
NheI and HindIII. The resulting expression plasmid was named pBH3093.
The His-tagged Fab fragment of SAR650984 (SAR650984-Fab) was produced by
transient transfection of light chain expression plasmid pBH3045 and heavy chain
expression plasmid pBH3093 in HEK293-FS™ cells. Purification was performed at day
10 from culture supernatant by affinity chromatography on IMAC (HisTrap, GE
Healthcare) using imidazole gradient in PBS. The pool of fractions containing
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SAR650984-Fab was purified by size exclusion chromatography using Superdex 200
(GE Healthcare) equilibrated with PBS.
Preparation of non-glycosylated soluble CD38
DNA encoding a signal peptide (MGWSCIILFVATATGVHS, coded by mouse Igh-VJ558
gene), followed by the huCD38 extracellular domain R45-I300 (UniProt: P28907) was
generated by a synthetic gene approach. Four mutations were introduced to prevent
potential N-glycosylation: N100D, N164A, N209D and N219D. In addition, a NheI and a
HindIII site were also added at 5’ and 3’ of the synthetic DNA fragment, respectively.
The fragment was cloned into a mammalian expression vector between the NheI and
HindIII sites to obtain the final expression vector pBH3133.
A non-glycosylated mutant of human CD38 (R45-I300) was produced by transient
transfection of expression vector pBH3133 into HEK293-FS™ cells. Ammonium
sulphate was added to the supernatant to a final concentration of 2 M and the pH was
adjusted to 7.6. This solution was loaded onto a Phenyl Sepharose HP column
equilibrated with 2M ammonium sulphate, 50 mM Tris-HCl, pH 7.6. The column was
washed with the same buffer and the bound protein was eluted with a linear gradient of
ammonium sulphate (from 2 to 0 M). The elution fractions containing non-glycosylatedCD38 were pooled and dialyzed against 50 mM sodium acetate buffer at pH 4.05. The
dialyzed sample was loaded onto a SP Sepharose HP column equilibrated with the
same buffer. The column was eluted with a linear salt gradient (0-1 M NaCl). Fractions
containing CD38 were pooled and purified on Superdex 200 (GE Healthcare).
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Structure determination of huCD38-SAR650984-Fab complex
Non-glycosylated-CD38 was mixed with the SAR650984 Fab fragment at 1.5 moles of
antigen per 1 mole of Fab and incubated for 30 minutes at room temperature. The
Antigen/Fab-complex was concentrated by spin-ultrafiltration and purified by SEC. All
purifications were monitored by measuring the UV absorbance at 280 nm. Eluted
fractions were analyzed by SDS-PAGE and analytical SEC. Protein concentration was
determined by using calculated epsilon absorbance of each protein or complex at 280
nm. Extensive crystallization trials were performed and crystals of this complex were
successfully grown in 45% PEG400, 0.1M 2-(N-morpholino) ethanesulfonic acid (Mes)
pH = 6 with the complex concentrated to 25 mg/mL. Crystals belonged to space group
P212121 and diffracted up to 1.53 Å on a synchrotron beamline. Free CD38 and a
chimeric model of Fab structures (pdb code 1s78 and 2osl) were used as models for
molecular replacement. One Fab and one CD38 were identified independently with
programs Phaser and Molrep (part of the CCP4 software suite, Oxon, U.K.). Structure of
CD38/SAR650984 was then refined with the program AutoBuster (Global Phasing Ltd,
Cambridge, U.K.) up to Rfac=18.8% and Rfree=20.8% at 1.53 Å resolution.
Competition experiments
SAR650984 and control huIgG1 were conjugated to Alexa Fluor® 488 (AF) using a kit
from Life Technologies and 5 and 6 fluorochromes/antibody molecule were obtained
respectively. SAR650984-AF and HB7-FITC (BD Biosciences) were incubated at
1.5 µg/mL in the presence of increasing concentrations of unlabeled SAR650984 with
3 x 105 Molp-8 cells/sample in PBS supplemented with 0.1% BSA and 5% human
serum for 30 min at 4°C. Samples were analyzed by flow cytometry on a MACSquant
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10 apparatus with the MACSquantify software (Miltenyi Biotec) for acquisition and
VenturiOne (Applied Cytometry) for analysis. Median fluorescence intensity (MFI) for
each data point was normalized as percent (%) binding against the MFI of isotype
antibody control (0% binding) and MFI in the absence of SAR650984 competition
(100% binding). Percent binding was plotted against the concentration of unlabeled
SAR650984 and sigmoidal dose-response curves were generated using GraphPad
Prism (GraphPad Software, San Diego).
Supplementary Table S1
Patient
Serum level of IgG
(mg/mL)
MM1
18.9
MM2
43.1
MM3
35.3
MM4
33.7
MM5
27.6
MM6
40.0
MM7
37.4
Supplementary Table S1. Serum level of IgG in multiple myeloma patient samples
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Supplementary Figure Legends
Supplementary Fig. S1. SAR650984 binding and activity on normal human blood cells.
SAR650984 binds to CD14+ monocytes (A) and CD3+ T cells (B) with an apparent Kd
of 0.2 nM. (C) CD38 expression on normal human blood cells by quantitative flow
cytometry. (D) Human blood cell depletion by SAR650984, rituximab and alemtuzumab
(average of n=2).
Supplementary Fig. S2. Close-up view of the salt-bridges interactions between
huCD38 (gray) and the heavy-chain of SAR650984-Fab (yellow). The figure is rotated
by approximately 90° along the plane axis with respect to Figure 4C. Four salt-bridges
are indicated in black and form the interactions between heavy chain loops H1 (D31)
and H2 (D55 and D57) of the SAR650984-Fab with Arg194 (R194) and Lys111 (K111)
of huCD38.
Supplementary Fig. S3. Simultaneous binding of SAR650984 and anti-CD38 antibody
HB7. Flow cytometry analysis of fluorochrome-labeled SAR650984 and HB7 antibody
binding to Molp-8 cells in the presence of increasing concentrations of unlabeled
SAR650984.
Supplementary Fig. S4. Overlay of CD38-SAR650984-Fab and CD38-HB7 (PDB ID
code 3RAJ). Surface and ribbon representation of HB7 heavy chains (dark pink), HB7
light chains (light pink), SAR60985-Fab heavy chains (yellow), SAR60985-Fab light
chains (green) and human CD38 (gray) are shown.
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