srf231, a fully human, high affinity anti-cd47 antibody ... · isotype srf231 isotype srf231 0.16...
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Isotype SRF231
Isotype SRF231
0.16
0.08nm
00 200 400 600 800
Time(s)1000 1200 1400
SRF231, a fully human, high affinity anti-CD47 antibody, exerts potent preclinical antitumor activity through engagement of the FcR, CD32aMarisa O. Peluso, Kshama A. Doshi, Caroline M. Armet, Li Zhang, Rachel W. O’Connor, Jamie Strand, Lilia Merbouche, Matthew Rausch, Jonathan A. Hill, Benjamin H. Lee, Pamela M. Holland, Vito J. Palombella, Alison M. PatersonSurface Oncology, Inc. Cambridge, MA, USA Presented at SITC2019 on November 9, 2019
Background
SRF231 is a High Affinity CD47 Antibody with Slow Off Rate andNo Agglutinating Properties
SRF231 Exerts Antitumor Activity Through Phagocytosis and Cell Death
Single-Dose Administration of SRF231 in the Raji Xenograft Model Leads to Sustained Antitumor Activity,Cytokine Induction, Macrophage Infiltration, and Tumor Necrosis
SRF231-Induced Antitumor Activity is Dependent on Fc Engagement of FcγRIIa (CD32a)
SRF231 Retains Antitumor Activity in Washout Conditions & Sub-Maximal Receptor Occupancy is Sufficient for Maximal Activity
Conclusions
• CD47 is a transmembrane protein that acts as a “Don’t Eat Me” signal to evade immune recognition
• CD47 is overexpressed in multiple cancer subtypes and is associated with poor prognosis
• Several anti-CD47 molecules designed to antagonize the CD47 axis are being tested in the clinic
• Preclinical characteristics and antitumor mechanisms of the investigational agent SRF231, a fully human antibody targeting CD47, are described
• SRF231 is a high affinity, CD47-targeting antibody that delivers an activating signal to myeloid cells via CD32a
• SRF231 is a high affinity, CD47-targeting antibody with no agglutinating properties• SRF231 elicits antitumor activity via apoptosis and phagocytosis involving macrophage engagement in a manner dependent on the FcγR, CD32a• SRF231 displays favorable preclinical characteristics regarding its RO/tumor exposure/efficacy relationship• SRF231 is currently being evaluated in a Phase I clinical trial [NCT03512340] in advanced solid tumors and lymphomas
Figure 1. SRF231-hCD47 binding kinetics assessed by biolayer interferometry. (A) SRF231 immobilized using anti-hu Fc biosensor surface; CD47-his used as analyte. Binding traces (top) and calculated interaction parameters (bottom). (B) Tumor cell lines and primary cells stained with fluorescently labeled SRF231 or isotype control. MFI values obtained by flow cytometry and data reported as the MFI relative to isotype control.
Figure 2. (A) RBCs from patients with hematologic cancers (n=3) incubated with CD47 mAbs overnight. Aggregation assessed by % doublet formation using flow cytometry. (B) Hu whole blood incubated with 100 μg/mL Ab for 1h. Smears imaged and analyzed qualitatively for evidence of RBC agglutination. (A and B) CD47 reference Abs included as positive (SRF4.2C11) and negative (2D3) controls. (C) Jurkat cells incubated with anti-CD47 mAbs for 72h and agglutination assessed by imaging. Anti-CD47 mAb (B6H12) was a positive control.
Figure 3. (A-F) CFSE-Jurkat targets cultured with hu monocyte-derived macrophages (MoDM) at a 9:1 T:E ratio and incubated with mAb for 2 or 24h at 37°C. Cells stained with anti-CD14 and viability dye and analyzed by flow cytometry. (A) Phagocytosis denoted by % CFSE+ tumor cells within CD14+ macrophages. (B) Tumor cell death denoted by % non-viable cells within non-phagocytosed target population. (C-F) Depletion of tumor cell population (arrow), relative to isotype at 2h (C and E) and 24h (D and F).
Figure 4. Jurkat phagocytosis assays with hu MoDM (T:E 2:1) cocultured for 2h at 37°C. (A) Comparison of SRF231 (whole hIgG4) vs. SRF231 F(ab’)2. (B) Analysis of SRF231 Fc variants. (C) CD32a-specific blocking Ab (IV.3) abrogates SRF231-mediated phagocytosis. (D) IV.3 abrogates SRF231-mediated tumor cell death within the phagocytosis assay.
Figure 5. (A and B) CFSE-Jurkat or Raji target cells co-cultured with macrophages in the presence of excess SRF231 or in "Washout" conditions where excess drug removed from target cells prior to coculture with hu MoDMs. Targets and macrophages co-cultured for 2h at 37°C. Phagocytosis reported as % CFSE+ target cells within CD14+ macrophages via flow cytometry. (C) Correlation between % Max Phagocytosis vs. % Receptor Occupancy (RO) reveals that sub-maximal RO is sufficient for maximal phagocytosis induction.
Figure 6. (A-C) SCID mice bearing 100-150 mm3 Raji tumors treated once with SRF231 or isotype ip. (A) Mean tumor volume (n=8/grp). (B) Total hIgG4 plasma concentration (n=3/grp/timepoint). (C) SRF231 tumor exposure analyzed by total hIgG IHC staining (n=3/grp/timepoint). (D) SCID mice inoculated with luc-MM1.S cells iv. with BLI values 1.5x106 – 6.0x106 photon/second treated with SRF231 or isotype (n=4/grp). BLI imaging conducted 2-days following drug treatment.
Figure 7. SCID mice bearing 100-150 mm3 Raji tumors were treated once with SRF231 or isotype. (A) After 96h, tumors (n=3/grp) collected and tumor lysates analyzed for expression of mu IP-10 and MCP-1. (B-C) Raji tumor sections from isotype (168 h) and SRF231 (96 h) treated mice (n=3/grp) stained for F4/80 or H&E. (B) Representative F4/80 staining (left panel: 1X ,10X) and quantification (right panel) of % F4/80+ expression. (C) Necrotic tumor area (%) determined by quantitative image analysis of H&E stained tumor sections. P-values calculated using an unpaired t-test.
CD47/SIRPα Blockade
SIRPα
SIRPα Signaling Phagocytic FunctionCytokine Production
DeathSignals
FcR Function
SRF231
MCP-1
Fc Receptor(CD32α)
Imbalance of “eat me/don’t eat me signals”
Clusters of CD47 and IgGs enhance affinity for Fc receptor
Recruitment of macrophages to tumor
CD47 Clusters
Active Cancer Cell Compromised Cancer Cells
Tumor-ResidentMacrophage (Scaffold)
Tumor-ResidentMacrophage
Property SRF231
Isotype hIgG4 (fully human)
CD47 Binding Affinity Kd (M) 6.2x10-10
On-rate ka (1/Ms) 2.3x10+05
Off-rate kdis (1/s) 1.7x10-04
hIgG4 SRF231
2D3 SRF4.2C11 hIgG4 SRF231
mIgG1 B6H12
A A
A
B
A B C D
C A
A B C
B C D
D
E
F
B C
B
CFSE103
103
0
0
104
104
105 1030 104 105
105
103
0
104
105
CD14
CD14
CFSE
CFSE103
103
0
0
104
104
105 1030 104 105
105
103
0
104
105
CD14
CD14
CFSE
Efficacy(Raji)
Efficacy A Cytokine Induction C NecrosisB Macrophage InfiltrationPlasma PK(hIgG4 ELISA)
Tumor Exposure(IgG IHC)
IP-100
100
200
300
400
500
Tum
or c
onc.
(pg/
mL)
Isotype 300 µg
SRF231 100 µg
** **
**: P<0.05
Isotype SRF231
1 x 1 x
10 x 10 x
0
5
10
15
F4/8
0+ (%
RO
I)
Isotype 100 µg
SRF231 100 µg
**
**: P<0.05
0
5
10
15
20
25Raji
% n
ecro
tic
tum
or a
rea
Isotype 100 µg
SRF231 100 µg
***
***: P<0.005
MCP-1
0.001 0.01 0.1 1 10 100
10000
20000
30000
40000
50000
SRF231 (µg/mL)
Rela
tive
MFI PBMC
CD34+
Bone Marrow
Raji
Jurkat
U937
MM.1S
Neutrophil
HL-60
OPM-2
Tumor Normal
0.0001 0.01 1 100 100000
10
20
30
40
Antibody (µg/mL)
% D
oubl
ets
hIgG4
SRF231
2D3
SRF4.2C11
0.001 0.01 0.1 1 10 1000
20
40
60
80
100Phagocytosis (2h)
Antibody (µg/mL)
%CF
SE+ (
of C
D14
+ )
SRF231IgG4
0.001 0.01 0.1 1 10 1000
10
20
30
40Cell Death (2h)
Antibody (µg/mL)
%Ce
ll D
eath
(non
-pha
gocy
tose
d Ju
rkat
s)
SRF231IgG4
0.001 0.01 0.1 1 10 1000
20
40
60
80
100Jurkat Cell Depletion (2h)
Antibody (µg/mL)
%N
on-P
hago
cyto
sed
Targ
ets
SRF231IgG4
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
Jurkat Cell Depletion (24h)
Antibody (µg/mL)
%N
on-P
hago
cyto
sed
Targ
ets
SRF231IgG4
SRF231 F(ab’)2
hIgG4
Antibody (µg/mL)
%CF
SE+ (
of C
D14
+ )
hIgGSRF231 (WT hIgG4)SRF231 (hIgG4; S288P, L235E)SRF231 (WT hIgG2)SRF231 (WT hIgG1)SRF231 (hIgG1; LLP-FES) SRF231 (hIgG1; N297A)
0
20
40
60
Antibody (µg/mL)
%CF
SE+ (
of C
D14
+ )
mIgG2b+SRF231CD32a(IV.3)+SRF231
hIgG4SRF231
0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 1000
5
10
15
20
Antibody (µg/mL)Dea
d (%
non-
phag
ocyt
osed
)
mIgG2b+SRF231CD32a(IV.3)+SRF231
hIgG4SRF231
Antibody (µg/mL)
%CF
SE+ (
of C
D14
+ )
0.001 0.01 0.1 1 10 1000
20
40
60
80
SRF231hIgG4
hIgG4 WashoutSRF231 Washout
0.001 0.01 0.1 1 10 1000
20
40
60
Antibody (µg/mL)
%CF
SE+ (
of C
D14
+ )
hIgG4SRF231hIgG4 WashoutSRF231 Washout
1 10 1000
20
40
60
80
100
%Receptor Occupancy
%M
ax P
hago
cyto
sis
RajiJurkat
0 10 20 300
500
1000
1500
2000
2500
Days after randomization
Tum
or v
olum
e (m
m3 )
Isotype controlSRF231 30 µg
SRF231 300 µgSRF231 100 µg
SRF231 administrationSRF231 10 µg
0.01
0.1
1
10
100
1000
Time (days)
SRF2
31 p
lasm
a co
nc (µ
g/m
L)
1 2 4 7
SRF231 300 µgSRF231 100 µg
21
SRF231 30 µgSRF231 10 µg
0
10
20
30
40
Time (days)
hIgG
4 po
siti
ve (%
)
Isotype control
SRF231 30 µgSRF231 100 µgSRF231 300 µg
1 2 4 7 21
SRF231 10 µg
0 1 20
1×106
2×106
3×106
4×106
5×106 (MM1.S-luc)
Days
BLI (
phot
ons/
seco
nd)
Isotype controlSRF231 100 µgSRF231 30 µgSRF231 10 µgSRF231 3 µg
Background BLI
P272
RajiJurkat
0.0001 0.001 0.01 0.1 1 10 1000
20
40
60
SRF231
Antibody (µg/mL)
%CF
SE+ (
of C
D14
+ )
0.001 0.01 0.1 1 10 1000
20
40
60
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