Post on 18-Jun-2020
transcript
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SRF231Isotype
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Antibody (µg/ml)0.0001 0.001 0.01 0.1 1 10 1000
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SRF231Isotype
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Cell Line EC50 Phagocytosis Index
Cancer
Jurkat 273 11 T-ALL
Raji 200 5 Burkitt
Toledo 241 32 DLBCL
OCI-Ly1 102 37
HL-60 1510 6 AML
MV4-11 1345 13
(ng/ml)
CD47 Monoclonal Antibody SRF231 is a Potent Inducer of Macrophage-Mediated Tumor Cell Phagocytosis and Reduces Tumor Burden in Murine Models of Hematologic Malignancies
Pamela M. Holland, PhD, Ammar Adam, DVM, Emmanuel Normant, PhD, Caroline M. Armet, Rachel W. O’Connor, Marisa O. Peluso, Andrew C. Lake, PhD, Jonathan A. Hill, PhD, Detlev Biniszkiewicz, PhD, Scott C. Chappel, PhD, Vito J. Palombella, PhD, Alison M. Paterson, PhD
1Surface Oncology, 215 First Street, Cambridge, MA 02142, USA
58th ASH Annual Meeting & Exposition | San Diego, CA | December 3-6, 2016
#1843
BACKGROUND• CD47 is a broadly expressed cell surface protein over-expressed by multiple tumor
types1-3 • CD47 has multiple binding partners (Figure 1) and negatively regulates phagocytosis
by interacting with the macrophage inhibitory receptor signal regulatory protein alpha (SIRPα)4,5
• Agents that block the CD47-SIRPα interaction have therapeutic potential in that they restore phagocytic uptake of CD47+ tumor cells in vitro and attenuate tumor growth in vivo
• Here we characterize SRF231, one of a panel of fully human CD47 antibodies, and demonstrate that it exhibits the desired criteria for clinical development
MYELOID CELLeg. MACROPHAGE
SRF231
INTEGRINS
SIRPα
THROMBOSPONDIN-1
FC RECEPTOR
CD47
TUMOR TARGET
Figure 1: CD47 regulates the interaction between tumor cells and macrophages. CD47 is a type I integral membrane protein with one extracellular immunoglobulin variable-like domain and five membrane-spanning segments4. CD47 takes part in several cis and trans interactions, including integrins, thrombospondin-1, SIRPγ (not shown) and SIRPα6. SIRPα is an inhibitory receptor that modulates several activating receptors, including Fc receptors5. Engagement of SIRPα by CD47 leads to inhibition of macrophage-mediated phagocytosis. By targeting CD47 with SRF231, phagocytosis against tumor cells can be restored.
SRF231 ANTIBODY GENERATION• Fully human anti-CD47 antibodies were generated using mice carrying human
variable heavy and light transgenes• Several antibodies were screened for desired characteristics, including no
hemagglutination or RBC phagocytosis; SRF231 was selected as the lead candidate
Ligand Analyte Kd (M) Assay Format
Human CD47-hFcSRF231 Fab 8.25E-10 Immobilized antigen +
SRF231SRF231 Full-length 1.10E-11
Table I: Biacore analysis of the interaction between SRF231 and human CD47On rate and off rate constants, for the Fab and full antibody of SRF231 binding to human CD47-Fc was measured by SPR. The affinity constant (Kd) of each interaction is shown.
Figure 2: SRF231 blocks the interaction between CD47 and SIRPα.Jurkat cells pre-treated with antibodies at increasing concentrations were washed and stained with bioti-nylated hu SIRPα and detected with SA-APC. Inhibition of SIRPα binding leads to decreased fluorescence intensity with increasing antibody concentration. B6H12 is a commercially available anti-CD47 antibody.
0.01 0.1 1 10 10010
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Antibody Concentration (μg/ml)
Isotype controlB6H12SRF231
SIRPα competition
Stre
ptav
idin
-APC
GeoM
ean
MULTIPLE MYELOMA CELLS ARE SENSITIVE TO SRF231 IN VITRO AND IN VIVO
MACROPHAGE ACCUMULATION AND SHIFT TOWARD M1 PHENOTYPE IN SRF231-TREATED TUMOR XENOGRAFTS
EFFECT OF MACROPHAGE DEPLETION ON SRF231 ANTI-TUMOR ACTIVITY
CONCLUSIONS• SRF231isahighaffinity,fullyhumanantibodyagainsthumanCD47
• SRF231promotesrobusttumorcellphagocytosisofhematologicalcelllinesandprimarytumorcellspreferentiallyoverTcellsorRBCin vitro
• SRF231showspotentin vivoanti-tumorefficacyinpreclinical modelsofAML,lymphomaandmultiplemyelomaasmonotherapyorincombinationsettings
• SRF231iscurrentlyinIND-enablingstudiesandisexpectedtoenterclinicaltrialsin2017
References1. Campbell IG, et al. (1992) Cancer Res 52, 5416–5420.2. Majeti R, et al. (2009) Cell 138(2), 286-299. 3. Chao MP,et al. (2010) Sci Transl Med 2(63), 63-94.
SRF231 BINDING CHARACTERISTICS • SRF231 binds with high affinity to human CD47 (Table I)• SRF231 is a potent blocker of the CD47-SIRPα interaction (Figure 2)
SRF231 PROMOTES PHAGOCYTOSIS OF HEME CANCER CELL LINES AND PRIMARY AML CELLS IN VITRO AND HAS ANTI-TUMOR ACTIVITY IN VIVO
SRF231 PREFERENTIALLY ENGULFS TUMOR CELL TARGETS AND DOES NOT ENHANCE PHAGOCYTOSIS OR AGGREGATION OF RBC
Figure 4: Enhanced phagocytosis is preferential for tumor cells over normal leukocytes and RBC. (A) Human macrophages were cultured 2 hr with CellTrace Violet-labeled Jurkat and CFSE-labeled normal T cells at 1:1 and analyzed by flow cytometry. The frequency of CellTrace Violet+ or CFSE+ targets remaining in the portion of cells that are CD14- (ie. cells that were not phagocytosed) is reported. Shown are mean values of technical replicates ± std dev. (B) Macrophages as in (A) were cultured 2 hr with CFSE-labeled human RBC and the indicated antibodies. Co-cultures were stained for CD14 and analyzed by flow cytometry. Phagocytosis was reported as % CD14+ macrophages that were CFSE+; doublets excluded. (C) Antibodies were incubated with 0.5% human RBC overnight. Aggregation is % of conjugates determined by analysis of side-scatter height vs. side-scatter width. ‘Positive control’ is in-house anti-CD47 clone that causes hemagglutination.
SRF231 COOPERATES WITH OPSONIZING ANTI-CD20 ANTIBODY IN VITRO AND IN VIVO
A
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Figure 8. Macrophages contribute to SRF231 mediated anti-tumor activity. SCID mice were inoculated with Raji cells s.c. When tumors reached 100-150 mm3, mice (n= 10/grp) were treated with 100 µg clodronate liposomes i.v. 3x/wk for 2 wks (blue arrows). One day following the second clodronate treatment, mice were treated i.p. with either 100 µg isotype control or SRF231 i.p. 3x/wk for 3 wks (black arrows). Data shown as mean tumor volumes ± SEM.
4. Brown E. (2001) Trends Cell Biol 11(3), 130-135. 5. Okazawa H, et al. (2005) J Immunol 174, 2004-2011.6. Oldenborg P. (2013) ISRN Hematol 2013, 614619.
Macrophages: F4/80
F4/80% of viable tumor area
%iNOS/%CD16310
%iN
OS /
%CD
163
Isotype d9 SRF231 d9
Macrophages: F4/80
F4/80% of viable tumor area
%iNOS/%CD16310
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Isotype d9 SRF231 d9
B
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F4/80% of viable tumor area
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Isotype d9 SRF231 d9
Macrophages: F4/80
F4/80% of viable tumor area
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Isotype d9 SRF231 d9
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SRF231 T cellIsotype T cellSRF231 JurkatIsotype Jurkat
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B6H12SRF231
Isotype
Human RBC Phagocytosis
% C
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Human RBC Aggregation
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0.039 µg/ml Rtx
SU-DHL-4
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SRF231 Rtx + SRF231 dose response
0.625 µg/ml Rtx
Raji
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Primary AML
MV4-11
Figure 5. Activity of SRF231 against multiple myeloma cells in vitro and in vivo. (A) Human macrophages were cultured for 2 hr with CFSE-labeled RPMI-8226, OPM2 or H929 target cells plus the indicated antibodies. Co-cultures were stained for CD14 and analyzed by flow cytometry. Phagocytosis reported as % CD14+ or MerTK+ macrophages that were CFSE+; doublets excluded. (B) SCID mice were inoculated with RPMI-8226, OPM-2 or H929 cells s.c. When tumors reached 100-150 mm3, mice (n= 6/grp) were treated with 100 µg SRF231 i.p. 3x/wk for 3 wks. Each line represents an individ-ual mouse. (C) CFSE-labeled bone marrow samples from multiple myeloma patients were incubated for 2 hr with macrophages and analyzed by flow cytometry as described in (A).
Figure 6: Enhanced SRF231-mediated phagocytosis in vitro and anti-tumor activity in vivo in the presence of CD20 opsonizing antibody. (A) Macrophages were cultured for 2 hr with CFSE-labeled Raji or SU-DHL-4 target cells plus the indicated antibodies. Co-cultures were stained for CD14 and analyzed by flow cytometry. Phagocytosis reported as % of CD14+ macrophages that were CFSE+; doublets excluded. Dotted green lines show maximal phagocy-tosis achieved with Rtx alone. (B) SCID mice were inoculated with Raji (left panel) or SU-DHL-4 (right panel) cells s.c. When tumors reached 100-150 mm3, mice (n= 10/grp) were treated i.p. with Isotype control, Rtx (5 mg/kg, 1x/wk for 3 wks), SRF231 (10 µg for Raji, 100 µg for SU-DHL-4, 3x/wk for 3 wks), or the combination. Data shown as median tumor volumes.
Figure 3: SRF231 promotes phagocytosis of hematologic tumor cell lines and primary AML cells in vitro and has anti-tumor activity in vivo. Human monocyte derived macrophages were cultured with the indicated CFSE-labeled tumor cell lines (A), or primary AML bone marrow cells (B). Cells were co-cultured for 2 hours in the presence of SRF231. Co-cultures were stained for CD14 and analyzed by flow cytometry. Phagocytosis reported as % CD14+ macrophages that were CFSE+; doublets excluded. ‘Phagocytosis index’ is the fold increase over isotype control at 10 µg/ml of antibody. (C) Nude mice were inoculated with MV4-11 cells s.c. Two days after implantation, mice (n= 10/grp) were treated with 30 or 100 μg SRF231 i.p. 2x/wk for 3 wks.
Figure 7. Analysis of macrophage accumulation by IHC staining of Raji xenograft tumors. SCID mice were inoculated with Raji cells s.c. When tumors reached with 100-150 mm3, mice (n = 10/grp) were treated with isotype control or up to 3 doses of 200 μg SRF231 i.p. on d2, d4 and d9. Tumors were collected on d2, d4 or d9 and analyzed by IHC. (A) Representative images of tumors stained with anti-F4/80. (B) Quantitation of F4/80 staining in Panel A. (C) Ratio of iNOS:CD163 positive staining in SRF231-treated tumors in viable tumor areas.