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Siglec-15 (S15), a member of sialic acid-binding immunoglobulin-
type lectins, is a highly conserved Type I cell surface protein, which
was previously reported to play a role in osteoclast differentiation and
bone remodeling1,2. Here we describe S15 as a novel co-inhibitory
ligand expressed on tumors and myeloid cells that suppresses T cell
function and promotes cancer growth. Blocking S15 by antibody
enhances anti-tumor immunity in preclinical models. 1 Macauley MS et al. Nat Rev Immunol. 2014 Oct;14(10):653-66.2 Hiruma Y et al. Bone. 2013 Mar;53(1):87-93.
Targeting Siglec-15 with NC318, a Novel Therapeutic Antibody to Enhance Anti-Tumor ImmunityLinda N. Liu1, Jun Wang2, Jingwei Sun2, Dallas Flies1, Chang Song1, Melissa Zarr1, Kristina Archer1, Alison McGuire1, Tom O’Neil1, Karla Maloveste1, Xinxin Nie2, Agedi Boto2,3,
Ron Copeland1, Sathya Janardhanan1, Tete Obot1, Jim Bingham1, Kevin N. Heller1, Sol Langermann1, Lieping Chen2
1NextCure Inc., Beltsville, Maryland, USA; 2Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, USA; 3Department of Pathology, Yale School of Medicine, New Haven, Connecticut, USA
BACKGROUND
SIGLEC-15 KNOCK OUT MICE
Figure 1: Myeloid-Lineage Cell Expression of S15 Inhibits Antigen-Specific
CD8+ OT-I T Cell Responses in VivoA: Brief outline of the study. B: The kinetics of OT-I T cell expansion and contraction in the
blood of WT, S15 KO and LysM-Cre S15 KO mice at indicated time points after OVA/poly
(I:C) immunization (n=4/group). Analysis shown is OT-I T cells as a percentage of total
CD8+ T cells. C: OT-I T cell proliferation in mouse spleens 5 days after transplantation.
Analysis shown is percentage of OT-I of total CD8+ T cells.
A B C
Figure 2: S15 Suppresses Anti-Tumor Immunity and Promotes GL261
Tumor GrowthA) GL261luc tumor growth in wild type or S15 KO mice post intracranial injection. B/C)
Flow cytometry analysis of tumor-infiltrating immune cells on Day 14 after GL261 tumor
inoculation. CD8+ T cells, CD4+ T cells, CD11b+ CD45high macrophages (MØ), CD11b+
CD45low microglia, and CD11c+ dendritic cells (DC) in total brain mononuclear cells were
quantified. B) Brain mononuclear cells were further re-stimulated with irradiated GL261-luc
cells for 5 days. C) Total number of IFN-γ-producing CD8+ T cells and CD4+ T cells was
determined based on live cell counting and intracellular cytokine staining. *P < 0.05.
Figure 3: S15 Suppresses Anti-Tumor Immunity and Promote B16.GMCSF
Tumor GrowthA & B) Subcutaneous B16.GMCSF tumor growth in S15 KO or C57Bl/6 wild type mice.
Tumor incidence and growth of individual mice (A) as well as percent survival (B).
Percentage of immune subsets (C) and T cells with effector phenotype (CD44hiCD62Llo) (D)
among total cells in the tumors from wild type or S15 KO mice. E) The % of IFN-γ and TNF-
α producing T-cells were shown based on intracellular staining of cells after ex vivo re-
stimulation with PMA and ionomycin.
SIGLEC-15 SUPRESSES T CELL IN VITRO
Figure 4: Siglec-15 Inhibits Antigen-Specific T Cell Responses In VitroA: Peritoneal macrophages from WT or S15 KO mice were harvested from C57Bl/6 wild
type or S15 KO mice peritoneal cavities. B: 293TKbOVA cells were transfected with S15
or control transfection with empty vector. Irradiated 293TKbOVA-S15+ or 293T-KbOVA-
control cells were co-cultured with purified OT-I T cells at 1:10 ratio. 3Hthymidine was
added to the co-culture two days later. The following day OT-I T cell proliferation was
measured by incorporation of 3H-thymidine. C: Supernatants from the cultured cells was
harvested prior to addition of 3H-thymidine and assessed for levels of IFN-γ and TNF-α by
ELISA.
Figure 5: Anti Siglec-15 (S15) Clone 5G12 Reverses S15 Fc Fusion Protein-
mediated Suppression on Human T cells. Human PBMCs from healthy donor were labeled with CFSE and added to anti CD3 coated
96-well plate plus S15 Fc fusion protein and indicated S15 mAbs. Three days later, the cells
were stained with anti CD4 and anti CD8 followed by FACS analysis of CFSElow cell
population. A) Human CD8 T cell proliferation; B) Human CD4 T cell proliferation
EFFECTS of 5G12 IN VIVO
➢ S15 immunosuppressive properties in the TME make it a rational target
for immunotherapy.
➢ NC318 is a high affinity humanized IgG1 mAb specific for S15 developed
to reverse tumor immune suppression and promote an effective anti-tumor
immune response.
➢ NextCure has completed IND-enabling studies and initiated evaluations of
NC318 in patients with advanced malignancies: “A Safety and
Tolerability Study of NC318 in Subjects With Advanced or Metastatic
Solid Tumor” https://clinicaltrials.gov/ct2/show/NCT03665285
A B C
ANTIBODY DEVELOPMENT
Siglec-15 KO
Human Siglec-15 Fc fusion
protein immunization
Confirmed high
serum titer via
ELISA
Screen clones
binding to cells
expressing human
or mouse Siglec-15
Hybridoma
generation
Confirm positive clones
binding to cells
expressing human or
mouse Siglec-15
Purified mAbs from
positive clones
mAb characterization:
affinity, binning
Test in vitro human
PBMC stimulation assay
Select top candidate
Humanization
No
C
D3
C
D3
al o
ne
Ct r
l F
c
S1
5. F
c
1B
2
1C
3
1C
12
1H
3
3H
10
5G
12
6F
8
8C
8
8H
8
10
G9
0
2 0
4 0
6 0
8 0
% o
f D
iv
id
ed
C
D8
+ T
c
ells
C D 3 + 5 g / m L S 1 5 . F c + 1 2 g / m L m A b
C D 3
*
No
C
D3
C
D3
al o
ne
Ct r
l F
c
S1
5. F
c
1B
2
1C
3
1C
12
1H
3
3H
10
5G
12
6F
8
8C
8
8H
8
10
G9
0
2 0
4 0
6 0
8 0
% o
f D
iv
id
ed
C
D4
+ T
c
ells
C D 3 + 5 g / m L S 1 5 . F c + 1 2 g / m L m A b
C D 3
*
A B
Clone
KD (nM)
(From 3 runs)
1H3 0.11 ± 0.02
5G12 0.30 ± 0.04
6F8 0.44 ± 0.08
10G9 0.55 ± 0.08
1C3 0.58 ± 0.13
3H10 0.60 ± 0.14
8C8 0.89 ± 0.25
8H8 1.30 ± 0.33
1C12 4.01 ± 1.05
1B2 4.33 ± 0.75
Table 1: Average kinetics values Table 2:Summary of Parent 5G12 and
NC318 Affinity Binding to Human Siglec-15
KD
(nM) R2
Kon
(1E+5/Ms)
Koff
(1E-4/s)
Parent
5G12 0.37 0.994 3.18 1.19
NC318 0.35 0.999 3.22 1.12
Figure 6: Binding of Anti-S15 Antibodies to Cells Expressing Mouse or
Human S15
(A) 293T.hS15 cells (NC318 EC50 = 2.42 nM) and (B) 293T.mS15 cells (5G12
parent EC50 = 1.26 nM).
A B
0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
2 9 3 T - h S 1 5
[ A n t i b o d y ] ( n M )
MF
I
(A
nt
ib
od
y
bi
nd
in
g
si
gn
al
)
N C 3 1 8
C o n t r o l m A b
5 G 1 2
0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
2 9 3 T - m S 1 5
[ A n t i b o d y ] ( n M )
MF
I
(A
nt
ib
od
y
bi
nd
in
g
si
gn
al
)
N C 3 1 8
5 G 1 2
C o n t r o l m A b
0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
2 9 3 T - h S 1 5
[ A n t i b o d y ] ( n M )
MF
I
(A
nt
ib
od
y
bi
nd
in
g
si
gn
al
)
N C 3 1 8
C o n t r o l m A b
5 G 1 2
0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
2 9 3 T - m S 1 5
[ A n t i b o d y ] ( n M )
MF
I
(A
nt
ib
od
y
bi
nd
in
g
si
gn
al
)
N C 3 1 8
5 G 1 2
C o n t r o l m A b
EFFECTS of NC318 IN VITRO
CONCLUSION
- 1 0 1 2
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
[ N C 3 1 8 ] L o g g / m LIL
-2
(p
g/m
L)
E C3 0
= 7 g / m L
D o n o r # 1
N C 3 1 8
C o n t r o l
- 1 0 1 2
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
[ N C 3 1 8 ] L o g g / m L
IL-2
(p
g/m
L)
E C3 0
= 1 5 g / m L
D o n o r # 2
N C 3 1 8
C o n t r o l
- 1 0 1 2
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
[ N C 3 1 8 ] L o g g / m L
IL-2
(p
g/m
L)
E C3 0
= 1 8 g / m L
D o n o r # 3
N C 3 1 8
C o n t r o l
- 1 0 1 2
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
7 0 0 0
[ N C 3 1 8 ] L o g g / m L
IL-2
(p
g/m
L)
E C3 0
= 7 g / m L
D o n o r # 4
N C 3 1 8
C o n t r o l
- 1 0 1 2
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
[ N C 3 1 8 ] L o g g / m L
IL-2
(p
g/m
L)
E C3 0
= 2 6 g / m L
D o n o r # 5
N C 3 1 8
C o n t r o l
Figure 10: NC318 Dose Dependently Stimulates Production of IL2 in Co-
Stimulated PBMC CulturesHuman PBMCs from healthy donors were added to anti-CD3 coated 96-well plate plus SEB
together with serially diluted NC318 or isotype control mAb. Supernatant was collected three
days later for IL-2 analysis.
Figure 7: 5G12 Reduces MC38.mS15 Tumor Lung Metastatic Nodules.A: Mice were treated with 5G12 on day 2 at 20 mg/kg, Q4D; B: Mice were treated with 5G12
on Day 3 at 3, 10 or 30 mg/kg, Q7D. **p<0.01
0 1 0 2 0 3 0
0
5 0 0
1 0 0 0
1 5 0 0
D a y s p o s t t u m o r i n o c u l a t i o n
Tu
mo
r V
olu
me
(m
m3
) C o n t r o l
5 G 1 2
a n t i P D - 1
C o m b o
* * * * * * * * * * *
* *
* * * *
* * * ** p < 0 . 0 5
* * p < 0 . 0 1
* * * * p < 0 . 0 0 0 1
A B
C
Na
i ve
Un
t re
at e
d
5G
12
P
D1
Co
mb
o
0
2
4
6
C D 8+
I F N +
T C e l l s
% o
f C
D8
+ C
ell
s
n=
3
n=
3
n=
2n
=2
n=
6
Na
i ve
Un
t re
at e
d
5G
12
P
D1
Co
mb
o
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
C D 8+
T N F +
T C e l l s
% o
f C
D8
+ C
ell
s
n=
3
n=
3
n=
2n
=2
n=
6
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0
0
2 0
4 0
6 0
8 0
1 0 0
D a y s p o s t t u m o r i n o c u l a t i o n
Su
rv
iva
l (
%)
C o n t r o l
5 G 1 2
a n t i P D 1
C o m b o
R e c h a l l e n g e
D a y 6 5 D a y 1 0 0
I L - 1 7 IP - 1 0 M C P - 1
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
3 0 0 0
4 0 0 0
C o n t r o l ( N = 1 0 )
5 G 1 2 ( N = 1 0 )
P D 1 ( N = 1 0 )
5 G 1 2 + P D 1 ( N = 1 0 )
N a i v e ( N = 8 )
pg
/mL
*
* *
* p < 0 . 0 5
* * p < 0 . 0 1
* * * p < 0 . 0 0 1
* *
* *
*
* * *
v s . C o n t r o l t r e a t e d
* *
*
D
Figure 8: 5G12 Monotherapy or in Combination with Anti-PD1 In
CT26/S15+ BMDM Tumor Model. A: Tumor volume; B: Kaplan-Meier survival plot; C: CT26-specific CD8+IFN-γ+ and
CD8+TNF-α+ T cells in mouse spleen collected on Day 107; D: Serum collected on Day 30 and
analyzed for 20 mouse cytokines (mouse 20-plex Luminex Kit from ThermoFisher).
2 6
0
2
4
6
8
I L - 6
ng
/mL
N a i v e
L P S + P B S
L P S + 5 G 1 2
H o u r s
2 6
0 . 0
0 . 5
1 . 0
1 . 5
T N F -
ng
/mL
N a i v e
L P S + P B S
L P S + 5 G 1 2
H o u r s
2 6
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
G M - C S F
pg
/mL
N a i v e
L P S + P B S
L P S + 5 G 1 2
H o u r s
2 6
0
5 0
1 0 0
1 5 0
2 0 0
I L - 1
pg
/mL
N a i v e
L P S + P B S
L P S + 5 G 1 2
H o u r s
2 6
0
2 0 0
4 0 0
6 0 0
8 0 0
I L - 1 8
pg
/mL
N a i v e
L P S + P B S
L P S + 5 G 1 2
H o u r s
Figure 9: 5G12 Significantly Increases LPS-Mediated Immune Activation in
Mice. Mice were first injected with PBS or 5G12 (10 mg/kg) and challenged with LPS 1h later.
Serum was collected 2h, 6h later and analyzed for pro-inflammatory cytokines.