Enhancement Of T-Cell Immunity To Osteosarcoma By Modulation Of

Programmed Death Receptor Pathway

Pooja Hingorani, Danielle Lussier, Joseph Blattman

Overview

• T cell tolerance and immune inhibitory pathways in osteosarcoma

• Review our data

Immune tolerance in osteosarcoma

• Loss of Class I HLA expression in osteosarcoma tumors related to worse overall survival*

• CTLA4 polymorphisms are associated with higher risk of developing osteosarcoma**

• Metastatic osteosarcoma expresses T cell Ig/mucin molecule 3 (TIM3), which in other tumor settings inhibits the function of infiltrating CTL, leading to tumor progression #

• B7-H3 expression, a co-inhibitory protein involved in tumor immune escape from T cells, inversely correlates with CTL infiltration in human osteosarcoma, and is indicative of poor prognosis in osteosarcoma patients @

*Tshukahara et al., Cancer Sci 2006; **Liu et al, DNA Cell Bio 2011; #Shang et al., Oncol Lett 2013; @ Wang et al., Plos One 2013

Relative expression of PDL1 in osteosarcoma

Shen J K et al. Cancer Immunol Res 2014;2:690-698

©2014 by American Association for Cancer Research

Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene expression.

Shen J K et al. Cancer Immunol Res 2014;2:690-698

©2014 by American Association for Cancer Research

Characterization of the origins of metastases.

Shen J K et al. Cancer Immunol Res 2014;2:690-698

©2014 by American Association for Cancer Research

Specific Aims• Evaluate expression of PD-L1 on tumor cells and PD-1 on TILs in patient

samples and metastatic osteosarcoma cell line (in vitro and in vivo)• Evaluate the functional ability of TILs infiltrating metastatic tumors• Evaluate the effect of PD-L1 blockade on development and progression of

pulmonary metastases in vivo• Evaluate the effect of combinational therapies (PD-L1 antibody +

chemotherapy; PD-L1 antibody + ipilimumab)

PD-L1 is expressed in human metastatic osteosarcoma

50um

50um50um

50um

Isotype

Metastatic Osteosarcoma

PD-L1

Primary Osteosarcoma50um

50um50um

50um

12/ 16 (75% ) of metastatic tumors had some PD-L1 expression

PD-1 is expressed on human metastatic TILs

50um

50um50um

50um 50um

50um50um

50um

50um

50um

Isotype

PD-1

50um

50um

50um

50um

Metastatic Osteosarcoma Primary Osteosarcoma

-13/16 (81%) of metastatic tumors had PD-1+ TILs-In 11/16 (70%) metastatic tumors, PD-1 expression on TILs correlated with PD-L1 expression on tumor cells

PD-L1 expression is increased in K7M2 osteosarcoma cells

PD-L1

% o

f Max

imum

40731848

Pre Implantation Post Tumor

30%

TILs from lung metastases are PD-1+

%PD

1+

SPLEEN CD8 TIL CD8

*

CD8

PD-1

SPLEEN TIL

94%

6% 87%

13%

Isotype

PD1

Healthy Lung

Late Disease Lung

Early Disease Lung 50 µm50 µm 50 µm

50 µm 50 µm 50 µm50 µm

TILs are functionally impaired in the presence of tumor cells

**

%IF

Nγ+

*

*

*

- + - + AgPD-L1+ K7M2

PD-L1- 4T1

%TN

F+

*

**

- + - + AgPD-L1+ K7M2

PD-L1- 4T1

%IL

-2+

**

- + - + AgPD-L1+ K7M2

PD-L1- 4T1

%IF

Nγ+

Control PD-L1 Ab

**

%TN

F+

**

*

Control PD-L1 Ab

%IL

-2+

**

*

Control PD-L1 Ab

A.

B.

C.PD-L1 blockade restores function to TILs

Blockade of PD-1:PD-L1 significantly enhances survival in metastatic osteosarcoma model

Days post implant

Perc

ent S

urvi

val

P=0.0005

30 day PD-L1 antibody treatment does not significantly improve survival compared to 15 day treatment

Is immune escape occurring?

PD-L1 antibody treatment changes inhibitory receptor expression

* *

PD-L1 antibody treated osteosarcoma is resistant to additional treatment when injected into naïve mice.

PD-L1

% o

f Max

imum

*

Combinational PD-L1 antibody treatment coupled with conventional chemotherapy

The combination improves survival as compared to chemotherapy alone but is similar to PD-L1 antibody alone.

Combinational PD-L1 antibody coupled with CTLA-4 antibody treatment improves survival

In conclusion

• PD-1/PD-L1 interaction induces immune tolerance in metastatic osteosarcoma

• PD-L1 blockade improves survival but not 100%• Resistance to therapy develops by up-regulation of alternative

immune escape pathways • Combinational immune therapies may have the highest

impact on disease control in metastatic osteosarcoma

• Joseph Blattman• Danielle Lussier• John Johnson• Lauren O’Neill• Lizbeth Nieves• Megan McAfee• Susan Holechek

• Paul Dickman• Patients and families

Acknowledgements