Some cancers are very resistant to immunotherapy. Here I talk about two of those, and speculate on the role of the tumor microenvironment in blocking productive anti-tumor immunity.

1. Immunomodulatory Targets in the Tumor
Microenvironment
ImVACS, 24 August 2015
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2. The Tumor Microenvironment (TME)
• The TME
- biophysical properties
- cellularity
• Why is it important?
- the link to metastasis
- the concept of zoning
• Example of targets and their potential for
combinations with immune checkpoints
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3. TME Biophysical Properties
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• ECM rich (collagen, HA)
• High intersitial pressure
• Poor penetrance (vascular,
interstitial space)
• Hypoxia
• Necrosis
• Acidic (Lactic acid)
Aberrant expression of cellular
mediators
- growth factors
- cytokines & chemokines
- tryptophan
- adenosine
figure courtesy Chris Thanos, Halozyme
cartoon of a pancreatic tumor

4. TME Cellularity – who lives here?
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- Cell populations: friend, foe or both?
- More friends than foes = the tumor is winning
- Why: immune subversion and evasion
Macrophage
- effector or TAM?
Lymphoctye
- Teffector or Treg?
- NK?
Fibroblast
- resident (activated)?
- CAF?
Myeloid lineage
- MDSC?
- iDC?
- monocytes?

5. Other Attributes: The TME ...
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• Is immunosuppressive
• Can confer therapy resistance
- anti-apoptotic niches (e.g. in bone marrow)
- refugia for cancer stems cells
- shelter from selective pressure (treatment resistance)
• Supports metastasis – resistance >>> metastasis
• Remodels metastatic niches (Ovarian cancer example)
• Is highly diverse – within indications, between stages of an
indication (early, advanced, metastasis), across tumor types,
between patients
• We currently underappreciate this compexity, meaning we are at a
primitive level in understanding this field

6. Pancreatic TME as an instructive example
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• The fibrotic environment (desmoplasia) limits biophysical exchage: extracellular/
interstitial fluid, gases, proteins, metabolites
• abundant CAFs, collagen, hyaluronic acid (brown: ECM component)
image courtesy of Chris Thanos, Halozyme
HA
Malignant cells
Cancer associated
fibroblasts
Immune cells

7. Why Focus on Pancreatic Cancer?
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• Huge unmet need:
Incidence and Mortality
- US: 46,000 diagnosed/yr; 39,000 deaths. Worldwide: x10 or more...
- Rising incidence: incidence and mortality may double by 2030.
- 90% of diagnoses are for the highly lethal ductal adenocarcinoma (PDAC)
- Pancreatic cancer is the only cancer with a 5-year OS rate in the single
digits (6%). Most patients (~80%) die the first year.
- If disease is localized, the 5-year OS is 22%.
- However, >50% of patients are diagnosed with disseminated disease,
having a 5-year OS of 2%.
• Some modest successes with vaccination attempts (GVAX etc) suggests that
inducing an immune responses is possible
• No to little success to date with immune checkpoint monotherapy

8. TME extracellular matrix (ECM) composition
and median survival in pancreatic cancer
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Halozyme investor day presentation
7 January 2015

9. A note regarding HA (hyaluronan)
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• A major component of ECM and a very large glycosaminoglycan (MW often
measured in the millions).
• HA is remarkably viscous in physiological fluids (10 mg/ml has a viscosity
5000x that of water) while retaining elastisity - this is why it is used, although
with limited success, in treating degraded joints.
• HA contributes to a variety of cancer cell activities including cell proliferation
and migration, and in the case of pancreatic cancer appears to act in part as
a physical barrier, creating a gated community with it's own, internal,
zoning bylaws in certain cancers, including pancreatic.
• At least 14 fourteen carcinoma types have elevated HA levels in the tumor
cells or the surrounding stroma or both.
• In ovarian cancer, the correlation between HA level and progression is
sufficient to support the use of HA concentration as a prognostic marker.

10. One more ... hyaluronidase treatment
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Halozyme investor day presentation
7 January 2015
• destroying the HA component of the ECM
with pegylated-HA allows therapeutics
better access to the tumor

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Pancreatic mets may phenocopy the 1o tumor
• At least in some pancreatic cancer patients the mets resemble the primary
tumor in ECM composition
Whatcott et al. 2015. Clin Cancer Res; 21(15); 3561–3568.

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What does this tell us?
• For encapsulated solid tumors like pancreatic cancer TME integrity is
fundamental to tumor health.
• Questions we should ask about this example:
- what happens molecularly when the tumor architecture crumbles?
- how does the immune system respond?
- how does the tumor crawl back to life?
• What additional targets can increase efficacy?
• What targets within the TME are broadly useful?
- Hyaluronidase only useful in certain indications with very high HA contect.
• Can we model the TME across indications or within diverse
indications?
• OK, moving on...

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Targets Within the TME
• Within the TME, local factors take control of the community:
tumor zoning bylaws
• These factors have diverse sources and enforce zoning in myriad ways
- all have immuno-
suppressive properties
- cells can both generate
and respond to these
factors
- several of these targets
are being aggressively
developed
• CSF-1
Mahoney, Rennert, Freeman NRDD 14: 561–584 (August 2015)

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Ovarian Cancer as a model for TME-remodeling
and metastasis
• Primary ovarian cancer is treated by debulking surgery, either preceded by
(neoadjuvant) or followed by platinum-based chemotherapy. 5 year OS is
~30%.
• At diagnosis, about two thirds of patients will have peritoneal metastases.
Peritoneal met bulk quickly surpasses primary tumor bulk, and tumor burden is
inversely associated with survival.
• Platinum-resistant recurrent and metastatic disease may be treated with a
different chemo-combo plus the anti-VEGF mAb bevacizumab (Phase 3).
• So a potential path forward in ovarian cancer is to control the return of mets
following therapy, i.e. can we change the zoning bylaws before the tumor
rebuilds?
• First we have to understand the peritoneal metastatic niche.

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Ovarian Mets as a TME model
Initial steps are understood, yielding both biology and targets: migration of
neoplastic and accessory cells, remodeling of ECM, and expansion of
vasculature
• Transit of seeding cells into the peritoneum can be passive, but is enhanced by
CXCR4, VEGF, LPA and VCAM-1.
• Engagement of mesothelial cell layer requires ECM proteins and CD44 (the HA
receptor) to anchor, and proteases to fragment fibronectin and vitronectin and allow
alpha integrin attachment: remodeling begins with those activated integrins...
• Alpha integrin activattion (by LPA etc) in turn activates TGFβ. TGFβ, VEGF, and diverse
chemokines cooperatively set up a proangiogenic and immuno-suppressive cascade.
• As immune cells respond, CD8+ T and NK cell secretion of IFNγ induces IDO
expression, further propagating immunosuppression.
• In the meantime, abundant CXCL12 and CSF-1 expression recruits immature myeloid
cells, fostering the development of the MDSC population
• Thus, Zoning Bylaws are established....

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Ovarian Cancer and immunotherapy
• However, some ovarian cancer patients can respond to immunotherapy, as
we have learned over the last few years:
• ipilimumab (anti-CTLA4, intermittent dosing) + GVAX in metastatic ovarian
cancer.
- ORR 9% (n = 11). Hodi et al. 2008. Proc Natl Acad Sci U S A. 2008: 105:3005-3010.
• nivolumab (anti-PD-1, 1 or 3 mpk q3w) in platinum-resistant ovarian cancer.
- ORR 23% (n = 13). J Clin Oncol 32:5s, 2014 (suppl; abstr 5511).
• avelumab (anti-PD-L1, 10mpk q2w) in recurrent or refractory ovarian cancer.
- ORR 10.5% (n=75). J Clin Oncol 33, 2015 (suppl; abstr 5509).
• pembrolizumab (anti-PD-1, 10mpk q2w) in PD-L1+ refractoy ovarian cancer.
- ORR = 11.5% (n=26). J Clin Oncol 33, 2015 (suppl; abstr 5510).

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Ovarian Metastases Illustrate One Type of Zoning
Some Ovarian cancers: Most Pancreatic cancers:
Active immunsuppression Gated community
Gajewski, Schreiber & Fu. 2013. Nat. Immunol. 14: 1014-1023.

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What should we look for....
• IHC can tell us if CD8+ T cells are present in a tumor section, and
perhaps some microenvironment data
• Additional useful information about the community and its zoning bylaws
might include:
- Immunosuppressive cellularity: Tregs, MDSC, TAM
- T cell responsiveness (IFNγ) and TCR clonality
- Immunosuppressive factors: IDO, adenosine, LPA
- resistance markers: CXCR4
- abundance of other targets e.g. VEGF
• What holds down responses in ovarian cancer?

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Immunosuppressive environment in ovarian cancer
• The TAM population (M2-type macrophages) is abundant and orchestrates
immunosuppression by secreting CCL21 and attracting Tregs. The presence
of Tregs is associated with poor prognosis and reduced OS in ovarian cancer.
• IDO is copiously produced by TAM/MDSC in ovarian cancer and regulates the
balance between T effector cells and Treg cells in favor of the Tregs, via
multiple mechanisms.
- Increased tumor burden (which entails the development of intratumoral hypoxia),
further drives IDO expression.
- High IDO expression correlates with poor outcome in ovarian cancer (also
endometrial, colon, melanoma, AML).
• PD-L1 expression on TAM, MDSC and tumor cells, and PD-1 expression on
T cells, is also associated with poor prognosis in ovarian cancer
• TAM can be depleted from the tumor microenvironment by blockade of the
CSF1R pathway.

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IDO on the horizon
target therapeutic developer partners summary
IDO INCB24360 Incyte AZN/
Medimmune
combo with MEDI4736 (anti-
PDL1), adv solid tumors including
pancreatic cancer
IDO INCB24360 Incyte BMS combo with nivolumab (anti-PD1),
adv solid tumors including ovarian
cancer
IDO INCB24360 Incyte Roche/
Genentech
combo with MPDL3280A (anti-
PDL1) for NSCLC
IDO INCB24360 Incyte Merck combo with MK-3475 (anti-PDL1)
in NSCLC and advanced solid
tumors
IDO &
TDO
F001287 Flexus BMS buy-out; combination regimens
with immunotherapies
IDO NLG919 NewLink Roche/
Genentech
combos with MPDL3280A (anti-
PDL1) and novel therapies

21. CIR 2015 in press
Salmonella – shIDO – PEGPH20, KPC orthotopic pancan model
with increased
proinflammatory
cytokines
produced

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Anti-CSF1R on the horizon
target therapeutic developer partners summary
anti-
CSF1R
FPA008 FivePrime BMS combo with nivolumab (anti-PD-1) in
6 cancer indications including
pancreatic cancer
anti-
CSF1R
emactuzumab Roche - combo with MPDL3280A (anti-PDL1)
in 5 cancer indications including
ovarian cancer
anti-
CSF1R
IMC-CS4 Eli Lilly - Phase 1 monotherapy: breast and
prostate cancers
CSF1R
inhibitor
JNJ-40346527 J&J - heme malignancies
CSF1R
inhibitor
PLX3397 Plexxikon/
Daaichi
Merck combo with pembrolizumab in
advanced solid tumors
CSF1R
inhibitor
BLZ945 Sloan Kettering
RI/Novartis
- ongoing?
CSF1R
inhibitor
AZD6495 Astra Zeneca - open innovation

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What can we expect
• Monotherapies will give no or modest response rates in these
difficult cancers
• Combo therapies require an understanding of complex mechanisms
employed in tumor defense, a defence that includes the TME
• For ovarian cancer there is clear hope, as the response rates
acheived, while low, offer a glimpse of what may be achieved in
combos
• For pancreatic cancer the challenge is to "tear down that wall" and
certainly agents like PEGPH20 are only part of the answer
• IDO and CSF1R are promising targets ... there are many others
• Finally...

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There is much to learn
stay tuned

25. Paul D Rennert
www.sugarconebiotech.com
rennertp@sugarconebiotech.com
on Twitter @PDRennert
1-508-282-6370 (USA)

26. www.sugarconebiotech.com
Backup slide: IDO combination with HA