A novel immunocytokine fusion protein combining tumor-targeting anti-CD47 antibody with GM-CSF cytokine for enhanced antitumor efficacy

Zhengyi Wang, Wei Cao, Taylor B. Guo, Lei Fang, and Jingwu Zang, I-MAB Biopharma Inc., Shanghai, China

ABSTRACT

METHODS

AACR 2018 #5622

CD47 blockade has emerged as a promising cancer immunotherapy by promoting phagocytosisof tumor cells. However, this treatment strategy may be limited by the predominantaccumulation of macrophages with pro-tumor M2 phenotype. Therefore, reprogramming ofM2-type macrophages into M1-type macrophages has been considered as a better potentialtherapy to boost the antitumoral effects by macrophages. The key myeloid cytokine GM-CSF isapproved as an adjunctive agent in the treatment of cancer by virtue of its ability to stimulatehematopoiesis and particularly induce the differentiation of pro-inflammatory M1 macrophages.Here we report the development of a novel immunocytokine composed of a newly discoveredCD47 IgG1 fused with GMCSF at the C-terminus (1F8-GMCSF). In macrophage-tumor cell co-culture, treatment with 1F8-GMCSF fusion molecule led to significantly higher levels of tumorcell phagocytosis and M1 characteristic cytokines production than that with anti-CD47 alone.1F8-GMCSF demonstrated superior efficacy in suppression of tumor growth as well as shift oftumor associated macrophages from M2 to M1 phenotype compared to 1F8 or GM-CSFtreatment alone or in combination. In vivo tracking of injected anti-CD47 antibodies by liveimaging confirmed the enriched localization of antibodies in the CD47+ tumor site. Takentogether, our data showed that 1F8-GMCSF fusion exhibited enhanced phagocytosis and M1macrophage activation, leading to the superior antitumor efficacy.

Generation of anti-CD47-GMCSF fusion molecule: A lead anti-CD47 mAb (1F8) was identifiedfrom panning of a naïve human library for binding to human CD47 extracellular domain (ECD).All binders with unique VH and VL were converted to full IgG and screening through a series offunctional assays. The human GMCSF sequence was then fused via various flexible linkers to theC-terminus of 1F8’s Fc to generate different formats of anti-CD47-GMCSF fusion molecules.

In vitro phagocytosis assay: Human PBMC-derived macrophages were seeded in 24-well plates(1 x 105 cells/well) overnight. On the next day, 5 x 105 CFSE-labeled Raji cells were added to themacrophages followed by addition of IgG control or anti-CD47-GMCSF fusion molecules.Phagocytosis activity was measured by analyzing the percentage of CFSE and CD14 double-positive cell population by flow cytometry.

TF-1 proliferation assay: Human erythroleukemic TF-1 cells were seeded at 1 x 104 cells/well ofa 96 well plate. Recombinant human GMCSF control or anti-CD47-GMCSF fusion was added tothe cells and incubated for 72 hours at 37°C. Cell proliferation was measured by adding CellTiter-Glo® according to the manufacturer’s recommendation.

Human M1 macrophage activation: Primary human macrophages were co-cultured with tumorcells in the presence of IgG control, GMCSF alone, anti-CD47 mAb alone or anti-CD47-GMCSFfusion for 24 hours. The culture supernatant was collected for the production of TNF-a, IL-6 andIL-12 and the cells were analyzed for the expression of CD80 by flow cytometry.

In vivo xenograft model and M1/M2 staining: NSG mice were subcutaneously engrafted withRaji cells and dosed twice a week with IgG control, 1F8 mAb, mouse GMCSF, 1F8 mAb + mouseGMCSF combo or 1F8-mouse GMCSF fusion (70 nmol per mouse) when the average tumorvolume reached 100-150 mm3. Tumors were excised and fixed in 4% paraformaldehyde forimmunofluorescence (IF). Sections were stained with anti-iNOS antibody for M1 macrophagesor anti-CD163 antibody for M2 macrophages and counterstained with DAPI for nuclei.

In vivo biodistribution: The biodistribution of anti-CD47 antibody was analyzed by in vivo liveimaging. 1F8 was labeled with indocyanine green (ICG) according to the manufacturer’s protocol(Dojindo Molecular Technologies) and intravenously injected into Raji cell-bearing mice (10mg/kg). At the time of injection and at 6, 24 and 72 hours after administration, mice wereimaged with IVIS Lumina III system to monitor the distribution of the ICG-1F8 antibody.

RESULTS

Figure 1. Design of anti-CD47-GMCSF Fusion (C4GM)

Promote tumor phagocytosis

Enhance anti-tumor immune responses

Retain the RBC-sparing properties

Maintain the potency of fusion cytokine

with the use of flexible linker

Trigger the shift of M2 to M1 phenotype

(potentiate phagocytosis)

Modulate the cytokine milieu of the

tumor microenvironment

Figure 2. C4GM demonstrated comparable potency in CD47 activity to the parental mAb.

ka (1/Ms) kd (1/s) KD (M)

1F8 3.61E+05 2.82E-03 7.81E-09

1F8-GMCSF 7.21E+05 4.43E-03 6.14E-09

1F8

IC50=1.6 nM

0.1 1 10 0.1 1 10 0.1 1 100

10

20

30

40

50

g/ml

IgG

1F8

1F8-GMCSF

% o

f C

FS

E+ c

ell

sl

in C

D14

+ M

A. CD47 binding

D. Phagocytosis by macrophage

1F8-GMCSF

B. SIRPa competition

IC50=1.3 nM

1F8

1F8-GMCSF

C. RBC agglutination

Figure 3. C4GM demonstrated comparable potency in GM-CSF activity to the recombinant protein.

-3 -2 -1 0 1 2 30.0

0.5

1.0

1.5

2.0

2.5 GMCSF

1F8-GMCSF

Normalized GMCSF [C]

Log (nM)

OD

450

-4 -2 0 20

2

4

6

Normalized GMCSF [C]

Log (nM)

Fo

ld c

han

ge o

f M

FI

-4 -3 -2 -1 0 10

50

100

150

Normalized GMCSF [C]Log (nM)

OD

450 (

X 1

04)

A. GMCSF receptor binding B. pSTAT5 signaling C. TF-1 proliferation

Figure 5. C4GM showed superior efficacy in Raji xenograft model with a shift of towards M1 polarization in TAMs.

CD47-Fab

IgG1 or IgG4 Fc

Human GM-CSFTumor tissue

0 3 6 9 12 15 180

500

1000

1500

2000 IgG (70 nmol, biw)

GMCSF (70 nmol, biw)

1F8 (70 nmol, biw)

1F8+GMCSF combo (70 nmol, biw)

1F8-GMCSF fusion (70 nmol, biw)

** ** **

Days post treatment

Tu

mo

r v

olu

me (

mm

3)

A. In vivo efficacy of C4GM in Raji model B. Shift of M1/M2 phenotype in TAMs

Figure 4. C4GM induced a robust M1 activation and enhanced phagocytosis in macrophage-tumor co-culture assay.

IL-6

0

2000

4000

6000

8000

**

IgG

1F8

GMCSF

1F8-GMCSF

pg

/ml

IL-12

0

200

400

600

pg

/ml

**

TNF-a

0

1000

2000

3000

4000

5000

pg

/ml

**

A. M1 activation in co-culture

CD80

0.0

0.5

1.0

1.5

2.0

2.5

fold

ch

an

ge o

f Ig

G c

on

tro

l

6.6 0.66 0.22 0.066 0.022 0.0060

2

4

6

8 IgG

1F8

GM-CSF

1F8-GMCSF

Concentration (nM)

fold

ch

an

ge o

f Ig

G c

on

tro

l

B. Enhanced phagocytosis in co-culture

1F8-GMCSF fusion induced higher levels of tumor phagocytosis at lower concentrations compared to 1F8 alone.

SUMMARY

Figure 6. Anti-CD47 Ab was enriched in tumor site after in vivo injection.

➢ C4GM is specifically designed to treat solid tumor with Mф infiltration – as opposed to CD47 mAb for hematological malignancies.

➢ C4GM demonstrates synergistic effects in enhancing M1 macrophage activation and tumor phagocytosis in vitro.

➢ C4GM shows superior efficacy in tumor growth inhibition in vivo compared to anti-CD47 alone or in combination with GMCSF, partly through focused local polarization of TAMs towards the M1 phenotype.

➢ Anti-CD47 mAb is highly enriched in tumor compared to other organs after systemic injection.

CD163 DAPIiNOS

IgG

1F8

GMCSF

Combo

Fusion

iNOS

IgG 1F8 GMCSF Combo Fusion0

5

10

15

iNO

S s

tain

ing

in

dex

CD163

IgG 1F8 GMCSF Combo Fusion0

5

10

15

CD

163 s

tain

ing

in

dex

D0 D1 D2 D3 D6

Do

rsal

Ven

tral

D1 D2 D3 D6

A. In vivo fluorescence imaging of CD47 Ab B. Enrichment of CD47 Ab in tumor

tumor

heart

liverlung

kidney

tumor heart liver lung kidney0.00

0.05

0.10

0.15

0.20

Co

nc

. o

f 1

F8

/tis

su

e (

g/g

)