1. Meredith Baker, MD PI: Bo Lu, MD – Radiation Oncology May 29th, 2014
The Role of Anti-PD1 Antibody in Combination with Radiation Therapy in an Orthotopic Lung Cancer Mouse Model
Meredith Baker, MD
PI: Bo Lu, MD – Radiation Oncology
May 29th, 2014

2. Lung Cancer
More people in the United States die from lung cancer than any other type of cancer
In 2010,
201,144 people in the United States were diagnosed with lung cancer
158,248 people in the United States died from lung cancer
5 year survival rates vary based upon stage at diagnosis, but overall range from 1-49%
More people in the United States die from lung cancer than any other type of cancer. In 2010, about 200,000 people were diagnosed and close to 160,000 people died from lung cancer. 5 year survival rates vary based upon stage of diagnosis, but overall, range from 1-49%

3. Antibody Therapeutics in Cancer
Over the past 16 years, the advent of “targeted”cancer therapies has emerged
This involves the use of drugs developed to inhibit oncogenic proteins or survival factors selectively expressed by tumors
There are currently 13 antibodies approved by the FDA for various oncologic indications
Hematologic cancers
Solid tumors - EGFR and HER2
Ipilimumab – CTLA4
Our approach to cancer therapy has remained fundamentally the same since the advent of chemotherapeutic drugs: surgery followed by sublethal administration of various cytotoxic compounds or radiation. However, over the past 16 years, the situation has started to change substantially with the advent of “targeted” cancer therapies: the use of drugs developed to inhibit oncogenic proteins or survival factors selectively expressed by tumors. Targeted agents involve the use of biotherapeutics and recombinant proteins (usually antibodies) that modulate targets expressed at the cancer cell surface.
There are currently 13 antibodies approved by the FDA for various oncologic indications
For example, treatment for hematologic cancers includes antibodies to B cell associated targets
Also, some antibodies are directed against targets expressed on solid tumors such as EGFR and HER2.
Ipilimumab: Blocks the activity of the negative regulator of T lymphocytes, CTLA4, resulting in the activation of the patient’s own immune response to cancer. This was the first clinically validated checkpoint pathway target.

4. PD-1 and PD-L1
So in discussing CTLA4, this leads us to PD-1 and PD-L1. Programmed death 1 (PD-1) protein is another T-cell coinhibitory receptor with a structure similar to that of CTLA4, but with a distinct biologic function and ligand specificity. PD-1 has two known ligands, PD-L1 and PD-L2. In contrast to CTLA4 ligands, PD-L1 is selectively expressed on many tumors, and on cells within the tumor microenvironment in response to inflammatory stimuli. Cancers where PD-L1 is selectively expressed include lung, breast, pancreatic, colorectal, bladder, HCC, RCC, head and neck, ovarian, cervical, brain, melanoma, prostate
This diagram, from a review article published in Science, demonstrates the checkpoint blockade and inhibition of immune suppression by anti-PD1. On the left, T cell influx into tumors results in the release of IFN-gamma, which up-regulates PD-L1 expression by tumor cells. PD-L1 binds to PD-1, which is expressed by activated T cells, where it can inhibit cytokine production and the cytolytic activity of PD-1+, tumor infiltrating cells, generating a negative signal that causes T cell exhaustion. Blockade of the interaction between PD-1 and PD-L1 potentiates immune responses, making PD-L1 a potentially promising target for cancer immunotherapy.
Sliwkowski, Mark X., and Ira Mellman.
Science (2013):

5. So, how can this be used in clinical practice
So, how can this be used in clinical practice? This is a study published in NEJM by Topalian et al (2012) in which they took patients with Melanoma, NSCLC, RCC, colon cancer and castration resistant prostate cancer. The majority of these patients had been heavily pretreated, with 47% receiving at least 3 prior regimens including chemotherapy and immunotherapeutics. These patients were treated with escalating doses of anti-PD1 antibody. Responses were assessed after each 8 week treatment cycle. Objective responses were seen in approximately 1 in 4-5 patients with NSCLC. As demonstrated in the figure above, this is from a patient demonstrating a partial response who received anti-PD1 Ab. Arrowheads show initial progression and then regression of his cancer. Although this article is a landmark study, in the interest of time, I’m only discussing a small portion of it. I highly recommend that you read it if you are interested.

6. Radiation Therapy
The combination of radiation therapy and immunotherapy holds particular promise as a strategy for cancer therapeutics
Radiation induced tumor-cell death as a potential source of tumor antigens for immunotherapy
Post-radiation tumor-cell modulation that allows more efficient immune-cell access
What is the role of radiation in this new class of immunotherapeutics?
Radiation therapy is conventionally used for local tumor control. Although local control of the primary tumor can usually prevent development of subsequent systemic metastases, tumor radiation fails to control pre-existing systemic disease, which may be present only as micrometastatic deposits. Combining radiation therapy with immunotherapy allows one to exploit 2 broad areas: a) radiation-
induced tumor-cell death as a potential source of tumor antigens for immunotherapy, and b) post-radiation tumor-cell modulation that allows more efficient immune-cell access and increased sensitivity to T-cell Killing.

7. Radiation Therapy
In lung cancer specifically, chemotherapy is not curative and radiation therapy provides locoregional control
So this diagram shows that the effect of radiation therapy is such that it increases antigen expression, which leads to increased T cell activation
Increased T cell activation leads to IFNγ production
Previous studies have demonstrated that IFNγ increases PD-L1 expression, but we needed to make sure that this applied to the murine model that we were using. And we also wanted to see if we could induce PD-1 expression, which hadn’t been shown before.

8. Hypothesis #1
Treatment of cells with radiation therapy will increase expression of PD-L1 and PD-1
So this led me to my first hypothesis this year, Treatment of cells with radiation therapy will increase expression of PD-L1 and PD-1

9. Effect of Radiation Therapy
Injected mice with Lewis lung carcinoma (LLC) cells
After 10 days, the tumors were treated with 25Gy radiation.
Three days post radiation therapy, spleen, lymph nodes and tumors were harvested.
To study irradiation, we injected B6 mice with 3x105 Lewis lung carcinoma cells, allowed tumors to develop for 10 days, and treated their tumors with 25Gy radiation. 3d post-RT, we harvested their spleens, LNs, tumors.

10. PD-L1 expression Effects of Radiation Therapy on PD-L1 Expression
No treatment
0.91%
0.54%
PD-L1 expression
IFNγ
77.6%
89.1%
Looking at our flow cytometry data, let me break it down for you. PD-L1 expression is on the vertical axis. The columns are two different lines of cancer cells, both LLC and ova-LLC, which is a more immunogenic cancer cell line. The cells were cultured with IFN gamma or treated with radiation therapy independently. Looking at the upper left corner of the flow diagrams, it is apparent that both IFN gamma and radiation therapy increases expression of PD-L1. Specifically, in the LLC cells (the first column), the no treatment group demonstrated ~1% expression of PD-L1, in comparison to the IFN-gamma group which showed that 77.6% of the cells expressed PD-L1 and 75.8% of the cells in the irradiated group expressed PD-L1.
20Gy
75.8%
70.6%

11. Effects of Radiation Therapy on PD-1 Expression
Lymph nodes
Tumor
1.71%
9.77%
PD-1
Post-irradiation (25Gy) No irradiation
40.8%
27.5%
In addition to the PD-L1 data, we looked at PD-1 expression after irradiation. Looking at the flow cytometry data, PD-1 expression is on the vertical axis. The columns are either lymph nodes or tumor cells, and the top row is no irradiation versus the bottom row of post-irradiation. Looking at the upper left hand corners, we see a significant increase in PD-1 expression in both LN cells and TILs. Specifically, looking at the LN no irradiation group, 1.7% of the cells demonstrated PD-1 expression, whereas 41% of the irradiated LN cells showed PD1 expression. To our knowledge, this was the first time demonstrating that radiation therapy induced PD-1 expression, not just PD-L1 expression.
(Black line shows cutoff based on positive controls, red line takes into account nonspecific staining)

12. Summary of evidence Radiotherapy increases tumor antigen expression
Increased antigen expression leads to increased T cell activation
Increased T cell activation leads to IFNγ production
IFNγ increases PD-L1 expression
Radiotherapy increases PD-L1 expression
Combining anti PD-1 mAb treatment and radiotherapy decreased tumor size in a triple negative breast cancer model
So what do we know so far?
And interestingly, in a previous study by Verbrugge et al (2012), they demonstrated that…combining anti PD-1…

13. Hypothesis #2
Adding anti-PD-1 antibody to a radiation therapy treatment regimen will augment tumor killing in an orthotopic lung cancer model
So this brings me to my second hypothesis this year:
Adding anti-PD1 antibody to a radiation therapy treatment regimen will augment tumor killing in an orthotopic lung cancer model.
As an aside, this is really the crux of the research being done in my lab. The future of combination immunotherapy and radiation therapy is very real and data is incredibly new, really only good results being published since early It’s novel and it is definitely promising. Having the opportunity to work on this was an excellent opportunity. But I digress…

14. Experimental Plan
Does combining PD-1 or PD-L1 inhibition with RT enhance anti-tumor treatment?
Black/6 mice are injected with 2x105 Lewis lung carcinoma cells into their flank (total volume 50μL).
Irradiate mice
Groups split into 12Gy and 20Gy
Add α-PD-1
Will inject PD-1 antibody at scheduled times
Monitor tumor burden
Assess changes in T cell activation
Collect spleen, LNs, tumor (TILs)
Isolate lymphocytes
Black/6 mice are injected with 2x105 Lewis lung carcinoma cells into their flank (total volume 50μL).
These mice were focally irradiated with either 12 or 20 Gy
During the course of the experiment, PD-1 antibody was injected at scheduled times
The tumor burden was monitored with caliper measurements
And at the end of the time course, the mice were sacrificed and spleen, LN and tumor cells were harvested. These cells were then treated to isolate lymphocytes and examine for PD-1 expression.

15. Tumor Growth Delay After Irradiation and Injection with Anti-PD-1 Antibody
Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 RT X
Ab (ug)
200
100
Time (d)
Tumor volume (mm3)
This is a tumor growth delay curve from one of our experiments combining radiation therapy with injection of anti-PD1-antibody. There were 5 groups of mice, 2 receiving 12Gy and 2 receiving 20Gy plus the control group, and each set receiving either a control injection of IgG or anti-PD1-Ab. Looking at the chart, I draw your focus to this light blue line, which is the combination of 20Gy and anti-PD-1 antibody. This group had the greatest response in tumor growth delay, demonstrating a clear relationship in tumor growth and combination radiotherapy and immunotherapy. Day 14 is where the data is statistically significant

16. Expression of PD-1 on CD8 cells after Irradiation and Injection of anti-PD1 antibody
12Gy + IgG
12Gy + G4
20Gy + IgG
20Gy + G4
0.73%
3.71%
PD-1
We were pretty happy with the results from the Tumor Growth Delay curve and wanted to assess the tissues for PD-1 expression. So we analyzed the tumor, LN and spleen cells with flow cytometry. I’ve included a sample slide for the tumor cells here.
Looking at the 20Gy + IgG and 20Gy + PD1 groups (*spacebar for animation*), you can see a mild increase in PD-1 expression 0.73% to 3.71% *spacebar for animation*. This increase is as predicted because of the irradiation.

17. Abscopal Effect of Radiation Therapy and Anti-PD-1 Antibody
Tumor Receiving
Direct Irradiation
Tumor Receiving
Abscopal Treatment
Tumor Volume
Knowing that the direct effects of radiation therapy in combination with immunotherapy were positive, we wanted to see if the radiation therapy and/or anti- PD-1 antibody would have an abscopal effect on the contralateral tumor. For those who don’t know, an abscopal effect is the effect of radiation therapy at a site distant to the area of irradiation. It comes from the Latin prefix “ab” meaning “away from” and the Greek word “skopos” meaning “target”
2x105 Lewis lung carcinoma cells are injected into each flank (total volume 50μL).
The right flank tumor received 20Gy of direct irradiation.
The left flank tumor received no irradiation, so the size reflects the abscopal effects of radiation and/or antibody therapy.
So if you look at the chart on the right, focusing on the dark blue line, you see that radiation therapy has a significant, but mild effect on the tumor not being directly irradiated and, even more interestingly, the combination of anti-PD-1 antibody with radiation therapy (light purple line) had an even greater abscopal effect than the tumors receiving radiation therapy alone.
Time post-irradiation (d)

18. Conclusions Radiation therapy induces PD-L1 and PD-1 expression
Combination anti-PD1 antibody and radiation therapy has an ablative effect on tumor growth
Combination anti-PD1 antibody and radiation therapy demonstrate abscopal effects in this murine model
So our conclusions show that Radiation therapy induces, not only, PD-L1, but also PD-1 expression
Combination anti-PD1 antibody with radiation therapy has an ablative effect on tumor growth
Combination anti-PD1 antibody and radiation therapy demonstrate abscopal effects in this murine model
As an aside, the crux of the research being done in my lab was the combination of radiotherapy and immunotherapy. The future of combination immunotherapy and radiation therapy is very real and data is incredibly new, really only good results being published since early It’s novel and it is definitely promising. Having the opportunity to work on this was really an interesting opportunity.

19. Thank You Bo Lu, MD, PhD Hannah Myers, MS Nathaniel Evans, III MD
Scott Cowan, MD

20. Works Cited
Brahmer, Julie R., et al. "Safety and Activity of Anti–PD-L1 Antibody in Patients with Advanced Cancer." N Engl J Med (2012): Web.
Sliwkowski, Mark X., and Ira Mellman. "Antibody Therapeutics in Cancer." Science (2013): Web.
Topalian, Suzanne L., et al. "Safety, Activity, and Immune Correlates of Anti–PD-1 Antibody in Cancer." N Engl J Med (2012): Web.
Verbrugge I et al. “Radiotherapy increases the permissiveness of established mammary tumors to rejection by immunomodulatory antibodies.” Cancer Res (2012):