a novel immunocytokine fusion protein combining …2019/10/30 · a novel immunocytokine fusion...
TRANSCRIPT
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
)