0.01 0.1 1 10 1000

10

20

30H929

SRF231Isotype

0.0001 0.001 0.01 0.1 1 10 1000

10

20

30OPM2

Antibody (µg/ml)0.0001 0.001 0.01 0.1 1 10 1000

10

20

30RPMI-8226

% C

FSE+ (

of M

erTK

+ Mac

s)

Antibody (µg/ml) Antibody (µg/ml)

% C

FSE+ (

of C

D14+ M

acs)

% C

FSE+ (

of C

D14+ M

acs)

0 20 40 600

1000

2000

3000

Days Post Randomization 0 20 40 60

0

500

1000

1500

2000

Days Post Randomization 0 20 40

0

500

1000

1500

2000

Days Post Randomization

Tum

or V

olum

e (m

m3 )

Primary MM

% C

FSE+ (

of M

erTK

+ Mac

s)

Tum

or V

olum

e (m

m3 )

Tum

or V

olum

e (m

m3 ) SRF231

Isotype

SRF231Isotype

0

6

8

10

2

4

Isotype SRF231

H929 OPM2RPMI-82260.001 0.01 0.1 1 10 1000

20

40

60

80

SRF231 (µg/ml)

% C

FSE+ (

of C

D14+ )

0

10

20

30

40

% C

FSE+ (

of C

D14+ )

Isotype SRF231

Iso SRF231

0 5 10 15 20 250

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400

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Days Post Implant

Tum

or V

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e (m

m3 )

Isotype SRF231 (30 µg)SRF231 (100 µg)

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

100

1000

10000

100000

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

0 20 40 600

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SRF231 + ClodronateSRF231ClodronateIsotype

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

%iN

OS /

%CD

163

Isotype d9 SRF231 d9

B

C

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

%iN

OS /

%CD

163

Isotype d9 SRF231 d9

0.001 0.01 0.1 1 10 1000

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Antibody (µg/ml))

(Non

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SRF231 T cellIsotype T cellSRF231 JurkatIsotype Jurkat

Jurkat Input %

T cell Input %

%Dy

e+ of C

D14-

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Antibody (µg/ml)

B6H12SRF231

Isotype

Human RBC Phagocytosis

% C

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of C

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SRF231Isotype

B6H12 Positive control

Human RBC Aggregation

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0.01 0.1 1 10 1000

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SE+ (

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0.039 µg/ml Rtx

SU-DHL-4

0.01 0.1 1 10 1000

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SRF231 Rtx + SRF231 dose response

0.625 µg/ml Rtx

Raji

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SE+ (

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SU-DHL-4Raji

A

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A

B C

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.