inhibition of novel immune-checkpoint · 9/26/2019 · pan cancer genome analysis revealed that...

T cell

TCR M HC-I

V ISTA IGSF11

tumor cells

TCR M HC-I

V ISTA IGSF11

T cell tumor cells

IGSF11 pathwayblockade

Targeting classical immune-checkpoints of the adaptive immunity has shown great therapeutic efficacy in oncology, but only in a limited fraction of patients. Therefore, identification of novel druggable tumor targets with immune suppressive function is of high priority in oncology research. Using our iOTarg genetic screening platform in a lung cancer model, that fails to respond to PD-L1 inhibition, we identified and further validated immunoglobulin superfamily member 11 (IGSF11) as an important immune-checkpoint target. IGSF11 is a type I transmembrane cell adhesion molecule that was recently described as a putative ligand of V-domain Ig suppressor of T cell activation (VISTA). Its expression is largely restricted to immune-privileged sites in healthy individuals, whereas it is frequently upregulated in cancer. Using phage display technology, we raised a diverse panel of fully human monoclonal antibodies (mAbs) against IGSF11 that exhibited improved immune lysis of tumor cells in in vitro cellular assays. Furthermore, knockout of Igsf11 in MC38 murine colon adenocarcinoma cells led to a significant reduction in tumor outgrowth upon s.c. implantation in C57BL/6 mice and a favourable reshaping of immune microenvironment. Pan cancer genome analysis revealed that high expression of IGSF11 in human cancer patients correlates with poor overall survival, especially in response to classical immune-checkpoint therapy. Overall, our data highlight that IGSF11 is a potent immune suppressive target on tumor cells and that IGSF11 blocking antibodies have the potential to become monotherapy treatment option for many solid cancer patients that are currently underserved with classical immune-checkpoint therapies.

→→ iOTarg screening platform identifies human IGSF11 as a tumor-expressed immune checkpoint target in a setting where PD-L1 inhibition is ineffective

→→ IGSF11 interacts with the established immune checkpoint receptor VISTA

→→ Novel anti-IGSF11 mAbs were raised that led to increased immune lysis of tumor cells in vitro

→→ CRISPR knockout of Igsf11 in murine MC38 tumors results in significant tumor growth retardation, along with reshaping of the intratumoral immune compartment

→→ Clinically, high IGSF11 expression overall correlates with poor prognosis in multiple solid tumor indications and is also associated with poor survival in response to standard immune-checkpoint therapies

→→ Taken together, IGSF11 is a compelling drug target for immunotherapy, especially in patients refractory to classical immune checkpoint blockade

IGSF11 interacts with VISTA.

→→ IGSF11 : VISTA interaction was observed in both orientation in biochemical assays

IGSF11 KO in MC38 tumor cells strongly reduces tumor outgrowth in vivo and reshapes the immune microenvironment.

→→ CRISPR KO of Igsf11 in MC38 leads to a dramatic tumor growth inhibition in vivo and reshapes the immune milieu.

Generation and characterisation of fully human anti-IGSF11 monoclonal antibodies.

→→ Novel target-specific and species cross-reactive IGSF11 binding antibodies were generated

→→ VISTA blocking and non-blocking IGSF11 antibodies were identified

- DMS -273 (IGSF11 siRNA )

- DMS -273 ( Ctrl . siRNA )

- M579 -A2 (IGSF11 siRNA )

- M579 -A2 ( Ctrl . siRNA )Isotype Ab ctrl

IGSF11 expression

Cells

(nor

m.)

20 pM 10 pM0

1,000

2,000

3,000

4,000

5,000

DMS273 + pan-T cells+ T cell engager (anti-CD3 x anti-EpCAM)

IFN-

ɣ [p

g/m

L]

T cell engager only

si-Ctrlsi-PD-L1si-IGSF11

**

**

T cell engager

si-Cell death

Mock-treated

si-Ctrl

si-PD-L1

si-IGSF11

0.0

0.2

0.4

0.6

0.8

M579-A2-luc + TIL-209

Tum

or ce

ll vi

abili

ty (n

orm

.)

**

Viability z score (− TIL)

Cyto

toxi

city

LO

ESS

scor

e (+

TIL

)

−4

−2

0

2

4

−4 −2 0 2 4

negative control PD-L1

IGSF11

arrayed siRNA library + / -

co-culture

Tumor cell line

Patient-derived TIL culture

Immunecheckpoint

Immune activator

Negativecontrol

Negativecontrol

(A) iOTarg screening overview

(B) Discovery of novel immune-checkpoints in lung cancer

(C) IGSF11 knockdown e�iciency in tumor cell lines

(D) IGSF11 knockdown in lung or melanoma tumor cells increases T cell activity and tumor lysis

mIGSF11 -mFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

hIGSF11 -hFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

mIGSF11 -mFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

hIGSF11 -hFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

IOMX-0242

IOMX-0168

HumanIGSF11

MouseIGSF11

iOmx anti-IGSF11 mAbsIGSF11

VISTA

0.001 0.01 0.1 1 10 100 10000

50

100

150

200

IgG [nM]

Rem

aini

ng IG

SF11

bin

ding

[%]

IOMX-0168 family

IOMX-0242 family

Isotype

IGSF11-specific and human/mouse cross-reactive

Monovalent affinities (Fab)

VISTA inhibition

(A) Discovery of unique fully human IGSF11 blocking antibodies

(B) Highly specific and species cross-reactive antibodies identified

(C) IGSF11 – VISTA interactioninhibitionby antibodies





IGSF11 expression

Cel

ls (n

orm

.)

20 pM 10 pM0

1,000

2,000

3,000

4,000

5,000


IFN-

ɣ [p

g/m

L]

T cell engager only


**

**

T cell engager

si-Cell death

Mock-treated

si-Ctrl

si-PD-L1

si-IGSF11

0.0

0.2

0.4

0.6

0.8

M579-A2-luc + TIL-209

Tum

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orm

.)

**


Cyto

toxi

city

LO

ESS

scor

e (+

TIL

)

−4

−2

0

2

4

−4 −2 0 2 4


IGSF11


co-culture

Tumor cell line


Immunecheckpoint

Immune activator

Negativecontrol

Negativecontrol





mIGSF11 -mFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

hIGSF11 -hFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

mIGSF11 -mFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

hIGSF11 -hFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

IOMX-0242

IOMX-0168

HumanIGSF11

MouseIGSF11


VISTA

0.001 0.01 0.1 1 10 100 10000

50

100

150

200

IgG [nM]

Rem

aini

ng IG

SF11

bin

ding

[%]

IOMX-0168 family

IOMX-0242 family

Isotype



VISTA inhibition




500 250 125 62,5 31,3 15,6 7,81Time (sec)

n m

0 200 400 600 800

0

0,1

0,2

u

500 250 125 62,5 31,3 15,6 7,81Time (sec)

n m

0 200 400 600 800

0

0,1

0,2

VISTA vs.

IGSF11

AHC

IGSF11 vs.

VISTA

AHC





IGSF11 expression

Cells

(nor

m.)

20 pM 10 pM0

1,000

2,000

3,000

4,000

5,000


IFN-

ɣ [p

g/m

L]

T cell engager only


**

**

T cell engager

si-Cell death

Mock-treated

si-Ctrl

si-PD-L1

si-IGSF11

0.0

0.2

0.4

0.6

0.8

M579-A2-luc + TIL-209

Tum

or ce

ll vi

abili

ty (n

orm

.)

**


Cyto

toxi

city

LO

ESS

scor

e (+

TIL

)

−4

−2

0

2

4

−4 −2 0 2 4


IGSF11


co-culture

Tumor cell line


Immunecheckpoint

Immune activator

Negativecontrol

Negativecontrol









IGSF11 expression

Cells

(nor

m.)

20 pM 10 pM0

1,000

2,000

3,000

4,000

5,000


IFN-

ɣ [p

g/m

L]

T cell engager only


**

**

T cell engager

si-Cell death

Mock-treated

si-Ctrl

si-PD-L1

si-IGSF11

0.0

0.2

0.4

0.6

0.8

M579-A2-luc + TIL-209

Tum

or ce

ll vi

abili

ty (n

orm

.)

**


Cyto

toxi

city

LO

ESS

scor

e (+

TIL

)

−4

−2

0

2

4

−4 −2 0 2 4


IGSF11


co-culture

Tumor cell line


Immunecheckpoint

Immune activator

Negativecontrol

Negativecontrol









IGSF11 expression

Cells

(nor

m.)

20 pM 10 pM0

1,000

2,000

3,000

4,000

5,000


IFN-

ɣ [p

g/m

L]

T cell engager only


**

**

T cell engager

si-Cell death

Mock-treated

si-Ctrl

si-PD-L1

si-IGSF11

0.0

0.2

0.4

0.6

0.8

M579-A2-luc + TIL-209

Tum

or ce

ll vi

abili

ty (n

orm

.)

**


Cyto

toxi

city

LO

ESS

scor

e (+

TIL

)

−4

−2

0

2

4

−4 −2 0 2 4


IGSF11


co-culture

Tumor cell line


Immunecheckpoint

Immune activator

Negativecontrol

Negativecontrol





mIGSF11 -mFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

hIGSF11 -hFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

mIGSF11 -mFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

hIGSF11 -hFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

IOMX-0242

IOMX-0168

HumanIGSF11

MouseIGSF11


VISTA

0.001 0.01 0.1 1 10 100 10000

50

100

150

200

IgG [nM]

Rem

aini

ng IG

SF11

bin

ding

[%]

IOMX-0168 family

IOMX-0242 family

Isotype



VISTA inhibition




Introduction

A iOTarg screening overview

A Discovery of unique fully human IGSF11 blocking antibodies

B Discovery of novel immune- checkpoints in lung cancer

B Highly specific and species cross-reactive antibodies identified

A

D IGSF11 knockdown in lung or melanoma tumor cells increases T cell activity and tumor lysis

C IGSF11 knockdown efficiency in tumor cell lines

Conclusion

Results

(A) Assay set up

(B) IGSF11 blockingAb IOMX-0168 mediates strong immune lysisof tumorcells

(C) Antibodyfunctionale ects are specificand requiretargetantigenbinding

0

2×106

4×106

6×106

8×106

1×107

1 10 100

IGSF11 competition assay- Tumor cell viability -

rec. human IGSF11 protein [µg/mL]

Tum

or ce

ll vi

abili

ty [R

LU]

00

50,000

100,000

150,000

200,000

1 10 100

IGSF11 competition assay- Residual antibody binding -


Bind

ing

to IG

SF11

+ ce

lls [M

FI]

+ IOMX-0168 + rh IGSF11 protein+ Isotype ctrl. Ab + rh IGSF11 protein

Tumor + T cells + BiTE + rh IGSF11 [max. conc.]Tumor + T cells + BiTETumor cellsTumor cells

0

Tumor cells

only

Tumor + T cells

only

+ Isotype Ig

G

+ anti-PDL1

+ IOMX-0168

+ IOMX-0242

0

2×106

4×106

6×106

Colo cells + pan T cells

Tum

or ce

ll vi

abili

ty(R

LU)

****

40 µg/ml

Q161,0

Q23,70

Q330,2

Q45,13

103

104

105

106

102

103

104

105CD3+

96,7

CD8+

CD4+

CD3+

2×106

4×106

6×106

8×106

1 10 100 1000

MDA-MB-231/IGSF11 cells + pan-T cells

IgG concentration [ug/mL]

Tum

or ce

ll vi

abili

ty[R

LU]

+ Isotype IgG+ IOMX-0168 (family)+ IOMX-0242 (family)+ anti-PD-L1

Tumor cells + BiTE + T cellsTumor cells + T cells

T cells onlyTumor cells only

Tumor T cell

T cell engager(BiTE)

CD3EPCA M

Lysis

102

103

104

105

0

200K

400K

600K

SSC-

H

C Antibody functional effects are specific and require target antigen binding

FIGURE 4

A Schematic representation of in vitro cellular assay setup employing cross-linking of tumor and polyclonal pan-T cells using bi-specific antibody tools. For this, pan-T cells were purified from peripheral blood of healthy donors using negative isolation technique. Flow cytometric analysis depicts the CD3, CD4 and CD8 composition within the T cell isolate.B Purified pan-T cells were co-cultured with MDA-MB-231/IGSF11 recombinant tumor cells or endogenously expressing Colo tumor cells in the presence of suboptimal dose of T cell engaging BiTE and IGSF11 blocking antibodies, as indicated. Isotype IgG and anti-PD-L1 antibodies were used as negative and positive controls, respectively. Tumor lysis was measured after 72 hours of co-culture using the CellTiter-Glo (CTG) viability assay.C To the above setup, increasing concentrations of recombinant human IGSF11 protein were added to the culture to neutralize antibody binding to the target cells, thus leading to a reduction of tumor cell lysis (left). Successful sequestration of anti-IGSF11 antibodies during the assay time was demonstrated by transfer of cell culture supernatant to fresh MDA-MB-231/IGSF11 and assessment of residual antibody binding in FACS. Shown are mean of triplicates ± SEM.

Functional anti-tumor effect of anti-IGSF11 antibodies requires immune cell contact.

→→ IGSF11’s suppressive activity requires immune cell contact that can be abrogated by IOMX-0168 antibody

T cell Tumor

VISTA IGSF11

TCR MHC

Apoptosis

IFNg / TNF /Perforin / Granzymes

0

2×106

4×106

6×106

1 10 100 1000 10000

Colo / T cell co-culture assay + IOMX-0168

IgG concentration [nM]

Tum

or v

iabi

lity

[RLU

]

IOMX-0168 only

IOMX-0168 + T cells

IOMX-0168 + T cell sups

Isotype IgG only

Isotype IgG + T cells

Isotype IgG + T cell sups

Colo only

Colo + T cells

Colo + T cell sups

T cells only

+ TCs

(A) Intrinsic vs. extrinsic immune resistance pathways (B) IOMX-0168-mediated tumor lysis is T cell-dependent

T cell Tumor

VISTA IGSF11

TCR MHC

Apoptosis

IFNg / TNF /Perforin / Granzymes

0

2×106

4×106

6×106

1 10 100 1000 10000

Colo / T cell co-culture assay + IOMX-0168

IgG concentration [nM]

Tum

or v

iabi

lity

[RLU

]

IOMX-0168 only

IOMX-0168 + T cells

IOMX-0168 + T cell sups

Isotype IgG only

Isotype IgG + T cells

Isotype IgG + T cell sups

Colo only

Colo + T cells

Colo + T cell sups

T cells only

+ TCs

(A) Intrinsic vs. extrinsic immune resistance pathways (B) IOMX-0168-mediated tumor lysis is T cell-dependent

B IOMX-0168-mediated tumor lysis is T cell-dependent

A Intrinsic vs. extrinsic immune resistance pathways

FIGURE 5

A Immune suppression by IGSF11 can be mediated via tumor intrinsic or tumor extrinsic resistance pathways. The latter relies on contact with T cells. B Colo tumor cells were cultured with either purified human panT cells or with supernatant of anti-CD3/anti-CD28 bead-activated T cells in the presence of anti-IGSF11 or isotype control IgGs at increasing concentrations. Tumor lysis was measured after 72 hours using CTG viability assay. A dose-dependent tumor cell lysis was only observed in the presence of purified T cells. Shown are triplicates ± SEM.

mIGSF11 -mFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

hIGSF11 -hFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

mIGSF11 -mFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

hIGSF11 -hFc

Time (sec)

n m

0 100 200 300 400 500

0

0,2

0,4

0,6

IOMX-0242

IOMX-0168

HumanIGSF11

MouseIGSF11


VISTA

0.001 0.01 0.1 1 10 100 10000

50

100

150

200

IgG [nM]

Rem

aini

ng IG

SF11

bin

ding

[%]

IOMX-0168 family

IOMX-0242 family

Isotype



VISTA inhibition




C IGSF11 – VISTA interaction inhibition by antibodies

FIGURE 3

A Antibody discovery workflow.B Anti-IGSF11 mAbs are human-mouse cross-reactive and highly specific. IgG antibodies were loaded on a human Fc capture dip-in biosensor and binding of IGSF11-Fc was tested at 1 µM. Binding to closest countertarget homologs (CXADR-Fc and VSIG1-Fc, tested in a similar way) was not detectable (data not shown). C Inhibition of the IGSF11-VISTA interaction. VISTA-Fc was immobilized on a Maxisorp ELISA plate. 200 nM IGSF11-His was pre-complexed with a dilution series of anti-IGSF11 mAbs and subsequently transferred to the immobilized VISTA-Fc. Remaining IGSF11-His binding was detected with an anti-HIS antibody conjugated with HRP. Inhibitory and non-inhibitory antibodies were selected for further functional characterization.

FIGURE 1

A Schematic representation of the iOTarg screen in lung cancer setting employing high throughput genetic knockdowns (~5,200 genes) in luciferase-positive H23 NSCLC cell line co-cultured with HLA-A2-restricted TIL culture, established from resected tumor of a lung adenocarcinoma patient.B Quadraplot representation of the screening result depicting the performance of individual gene knockdowns on T cell-mediated tumor lysis (cytotoxicity score) on Y-axis and impact on cellular viability per se on X-axis. Controls are highlighted. C Surface expression of IGSF11 on DMS-273 (lung cancer) and M579-A2 (melanoma) cell lines was downregulated via lipid-based transfection of cells with target-specific siRNAs (or control siRNA) for 72 hours, followed by flow cytometry analysis.D Indicated siRNA-treated DMS-273 (left) or M579-A2 (right) tumor cells were cultured with human pan-T cells and a suboptimal dose of anti-CD3 x anti-EpCAM bispecific scFv (left) or patient-derived TIL209 (right). After 72 hours, IFN-γ ELISA (left) or tumor cell viability (right) was measured. Shown are triplicates ± SEM.

Inhibition of novel immune-checkpoint IGSF11 mediates efficient tumor cell killing in vitro and in vivo

Maximilian Aigner1, Sabrina Genssler1, Anchana Rathinasamy2, Stefanie Urlinger1, Jonas Zantow1, Stefan Bissinger1, Tillmann Michels1, Simone Braendle1, Ronny Milde1, Jörg Regula1, Apollon Papadimitriou1, Philipp Beckhove2 and Nisit Khandelwal1

1 iOmx Therapeutics, Martinsried/Munich, Germany2 Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany

Research Authors and affiliations

iOTarg screen identifies IGSF11 as a potent immune-checkpoint target on tumor cells.

→→ IGSF11 was identified as immune-checkpoint target on lung tumor cells via iOTarg screen

→→ Knockdown of IGSF11 resulted in improved immune lysis of various tumor cells

→→ Khandelwal N et al. (2015). EMBO Molecular Medicine 7, 450-463

→→ Mehta N et al. (2019). Cell Rep 28, 2509-2516→→ Van Allen EM et al. (2015). Science 350, 207-2011→→ Miao D et al. (2018). Science 359, 801–806

References DownloadFIGURE 2

A Human VISTA-Fc or human IGSF11-Fc was immobilized on an anti-human Fc dip-in biosensor. After a quenching step with human Fc fragments, the heterophilic interaction was measured at multiple concentrations. The heterophilic VISTA-IGSF11 interaction was detectable in both orientations, whereas a homophilic interaction of VISTA or IGSF11 was not detectable (data not shown).

(A) Assay set up



0

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Bind

ing

to IG

SF11

+ ce

lls [M

FI]



0

Tumor cells

only

Tumor + T cells

only

+ Isotype Ig

G

+ anti-PDL1

+ IOMX-0168

+ IOMX-0242

0

2×106

4×106

6×106


Tum

or ce

ll vi

abili

ty(R

LU)

****

40 µg/ml

Q161,0

Q23,70

Q330,2

Q45,13

103

104

105

106

102

103

104

105CD3+

96,7

CD8+

CD4+

CD3+

2×106

4×106

6×106

8×106

1 10 100 1000



Tum

or ce

ll vi

abili

ty[R

LU]




Tumor T cell


CD3EPCA M

Lysis

102

103

104

105

0

200K

400K

600K

SSC-

H

B IGSF11 blocking Ab IOMX-0168 mediates strong immune lysis of tumor cells

7 100 3 2015

MC38(s.c. )

Tumor growth

C57BL/6 n= 10/group

Flow cytometry

IGSF11 KO retards tumor growth in vivo and favours anti-tumorimmune milieu

(A) (B)

MC38-IGSF11 KO

0 8 11 14 18 200

250

500

750

1000

Days

Tum

or v

olum

e (m

m3 ) MC38-ctrl.

******

73% TGI

Ctrl. KO

15

20

25

30

gMDSCs(CD11b+ Ly6G+)

% o

f tum

or c

ells *

Ctrl. KO0

2

4

6

8

10

cytotoxic CD8+ T cells

% o

f tum

or c

ells

Ctrl. KO0

5

10

15

20CD3+ T cells

% o

f liv

ing

cells p=0.09

Ctrl. KO0

1

2

3CD4

+ T cells

% o

f liv

ing

cells

*

7 100 3 2015

MC38(s.c. )

Tumor growth

C57BL/6 n= 10/group

Flow cytometry


(A) (B)

MC38-IGSF11 KO

0 8 11 14 18 200

250

500

750

1000

Days

Tum

or v

olum

e (m

m3 ) MC38-ctrl.

******

73% TGI

Ctrl. KO

15

20

25

30


% o

f tum

or c

ells *

Ctrl. KO0

2

4

6

8

10


% o

f tum

or c

ells

Ctrl. KO0

5

10

15

20CD3+ T cells

% o

f liv

ing

cells p=0.09

Ctrl. KO0

1

2

3CD4

+ T cells

% o

f liv

ing

cells

*

A

IGSF11 KO retards tumor growth in vivo and favours anti-tumor immune milieu

A

B

FIGURE 7

A Association of cancer patient survival (TCGA datasets) with IGSF11 expression was checked using the TIMER (Tumor IMmune Estimation Resource) database. Kaplan-Meier survival curves were generated by separating patients in the top and lowest 30 % (33rd quantile) expression levels, respectively. p-values were calculated using the log-rank test.B Association of IGSF11 expression (pre-treatment) and patient survival after immune checkpoint blockade (PD-1 or CTLA-4) was checked using the TIDE (Tumor Immune Dysfunction and Exclusion) database. Cohorts of kidney cancer patients treated with anti-PD-1 (33 patients, Miao 2018) and melanoma patients treated with anti-CTLA-4 (42 patients, van Allen 2015) were analysed. Optimal cut-off values for IGSF11 expression were calculated by the database.

Progression-free survival (day)

Cum

ulat

ive

Surv

ival

Cum

ulat

ive

Surv

ival

IGSF11 expression level

Low (Bottom 30%)

High (Top 30%)

Time to Follow−Up (months)

log−rank P = 0.001

HNSCC

0.00

0.25

0.50

0.75

1.00 log−rank P = 0.001

0.4

0.6

0.8

1.0log−rank P = 0.032

0 50 100 150 200 250

0.25

0.50

0.75

1.00

Lung adenocarcinoma

HNSCC

Stomach adenocarcinoma

HNSCC

0 50 100 150 0 50 100 150

0 200 400 600 800

PD-1 blockade (Kidney cancer, Miao 2018)

IGSF11 high ( Top 25%)IGSF11 low (Bottom 75%)

log-rank p = 0.00146

0 200 600 1000 1400

log-rank p= 0.141

IGSF11 high (Top 33%)IGSF11 low (Bottom 66%)

CTLA-4 blockade (Melanoma, van Allen 2015)

0.4

0.2

0.0

0.6

0.8

1.0

0.4

0.2

0.0

0.6

0.8

1.0

High IGSF11 expression correlates with poor prognosis, especially in response to anti-PD-1/CTLA-4

(A)

(B)

Progression-free survival (day)

Cum

ulat

ive

Surv

ival

Cum

ulat

ive

Surv

ival

IGSF11 expression level

Low (Bottom 30%)

High (Top 30%)

Time to Follow−Up (months)

log−rank P = 0.001

HNSCC

0.00

0.25

0.50

0.75

1.00 log−rank P = 0.001

0.4

0.6

0.8

1.0log−rank P = 0.032

0 50 100 150 200 250

0.25

0.50

0.75

1.00

Lung adenocarcinoma

HNSCC

Stomach adenocarcinoma

HNSCC

0 50 100 150 0 50 100 150

0 200 400 600 800

PD-1 blockade (Kidney cancer, Miao 2018)

IGSF11 high ( Top 25%)IGSF11 low (Bottom 75%)

log-rank p = 0.00146

0 200 600 1000 1400

log-rank p= 0.141

IGSF11 high (Top 33%)IGSF11 low (Bottom 66%)

CTLA-4 blockade (Melanoma, van Allen 2015)

0.4

0.2

0.0

0.6

0.8

1.0

0.4

0.2

0.0

0.6

0.8

1.0

High IGSF11 expression correlates with poor prognosis, especially in response to anti-PD-1/CTLA-4

(A)

(B)

High IGSF11 expression correlates with poor survival in cancer patients, especially in response to classical immune-checkpoint therapies.

→→ IGSF11-high expressing patients show poor overall survival→→ IGSF11 potentially mediates further resistance to anti-PD1 or anti-

CTLA-4 therapies

FIGURE 6

A MC38-IGSF11 KO cells were generated by CRISPR-based knockout of Igsf11 in MC38 cells and clonal indels were verified by NGS. In vivo tumor growth kinetics of 1x105 MC38 ctrl. and MC38-IGSF11 KO tumor cells implanted s.c. in C57BL/6 mice.B Flow cytometry analysis of immune infiltrates of 1x106 MC38 ctrl. and MC38-IGSF11 KO tumors implanted s.c. in C57BL/6 mice at day 15. Mean ± SEM are depicted.

For tumor growth kinetics statistical analysis was calculated using a two-way ANOVA including Tukeys multiple comparison analysis. For statistical analysis of the immune profile a non-parametric Wilcoxon-Mann-Whitney Test with Welchs correction was used. Statistical significance is indicated as: * p<0.05, ** p<0.01, *** p<0.001

7 100 3 2015

MC38(s.c. )

Tumor growth

C57BL/6 n= 10/group

Flow cytometry


(A) (B)

MC38-IGSF11 KO

0 8 11 14 18 200

250

500

750

1000

Days

Tum

or v

olum

e (m

m3 ) MC38-ctrl.

******

73% TGI

Ctrl. KO

15

20

25

30


% o

f tum

or c

ells *

Ctrl. KO0

2

4

6

8

10


% o

f tum

or c

ells

Ctrl. KO0

5

10

15

20CD3+ T cells

% o

f liv

ing

cells p=0.09

Ctrl. KO0

1

2

3CD4

+ T cells

% o

f liv

ing

cells

*

B

High IGSF11 expression correlates with poor survival in cancer patients, especially in response to classical immune-checkpoint therapies.

(A) Assay set up



0

2×106

4×106

6×106

8×106

1×107

1 10 100



Tum

or ce

ll vi

abili

ty [R

LU]

00

50,000

100,000

150,000

200,000

1 10 100



Bind

ing

to IG

SF11

+ ce

lls [M

FI]



0

Tumor cells

only

Tumor + T cells

only

+ Isotype Ig

G

+ anti-PDL1

+ IOMX-0168

+ IOMX-0242

0

2×106

4×106

6×106


Tum

or ce

ll vi

abili

ty(R

LU)

****

40 µg/ml

Q161,0

Q23,70

Q330,2

Q45,13

103

104

105

106

102

103

104

105CD3+

96,7

CD8+

CD4+

CD3+

2×106

4×106

6×106

8×106

1 10 100 1000



Tum

or ce

ll vi

abili

ty[R

LU]




Tumor T cell


CD3EPCA M

Lysis

102

103

104

105

0

200K

400K

600K

SSC-

H

(A) Assay set up



0

2×106

4×106

6×106

8×106

1×107

1 10 100



Tum

or ce

ll vi

abili

ty [R

LU]

00

50,000

100,000

150,000

200,000

1 10 100



Bind

ing

to IG

SF11

+ ce

lls [M

FI]



0

Tumor cells

only

Tumor + T cells

only

+ Isotype Ig

G

+ anti-PDL1

+ IOMX-0168

+ IOMX-0242

0

2×106

4×106

6×106


Tum

or ce

ll vi

abili

ty(R

LU)

****

40 µg/ml

Q161,0

Q23,70

Q330,2

Q45,13

103

104

105

106

102

103

104

105CD3+

96,7

CD8+

CD4+

CD3+

2×106

4×106

6×106

8×106

1 10 100 1000



Tum

or ce

ll vi

abili

ty[R

LU]




Tumor T cell


CD3EPCA M

Lysis

102

103

104

105

0

200K

400K

600K

SSC-

H

IGSF11 blocking antibodies mediate immune lysis of tumor cells in an epitope-dependent manner.

→→ Novel IGSF11 blocking antibody from epitope class of IOMX-0168 mediate strong tumor lysis by immune cells

→→ Functional effect of antibodies were target-binding specific

A Assay set up

7 100 3 2015

MC38(s.c. )

Tumor growth

C57BL/6 n= 10/group

Flow cytometry


(A) (B)

MC38-IGSF11 KO

0 8 11 14 18 200

250

500

750

1000

Days

Tum

or v

olum

e (m

m3 ) MC38-ctrl.

******

73% TGI

Ctrl. KO

15

20

25

30


% o

f tum

or c

ells *

Ctrl. KO0

2

4

6

8

10


% o

f tum

or c

ells

Ctrl. KO0

5

10

15

20CD3+ T cells

% o

f liv

ing

cells p=0.09

Ctrl. KO0

1

2

3CD4

+ T cells

% o

f liv

ing

cells

*

inhibition of novel immune-checkpoint · 9/26/2019 · pan cancer genome analysis revealed that...

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