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.