Notes on Hypoxia and Immunotherapy Resistance

Introduction to Mike Curran's Work

Mike Curran is an associate professor of immunology at MD Anderson in Houston, Texas.
He completed his PhD at Stanford and pursued postdoctoral studies with Jim Allison.
Curran's work includes seminal research on combinations of immune checkpoint blocking antibodies (anti-PD-1 and anti-CTLA-4).
He has been recognized by SITC for his work on costimulatory molecules like anti-4-1BB antibodies.
Current research focuses on strategies to address cold tumors (e.g., pancreatic and prostate cancers) by increasing T cell infiltration and responsiveness to immunotherapy.

Overcoming Immune Suppression and Resistance to Immunotherapy

Curran's group aims to understand and overcome immune suppression and resistance to immunotherapy.
The lecture discusses ongoing work on hypoxia and its effects on immune suppression and resistance to immunotherapy.

Checkpoint Blockade and Cancer Immunotherapy

Checkpoint blockade can relieve off-switches on T cells, restoring tumor immunity.
Blocking CTLA-4 and PD-1, along with PD-L1 and B7-1, can improve outcomes.
Checkpoint blockade was a major innovation recognized with the Nobel Prize.
The translation of mouse studies to human patients was a key factor.
Metastatic melanoma saw significant survival improvements with CTLA-4 and PD-1 blockade, increasing from 18% to 80% two-year survival rates.
This combination became the first FDA-approved checkpoint combination.
High levels of immune-related adverse events remain a challenge.

Challenges in Immunotherapy for Cold Cancers

The majority of patients have cold cancers with low tumor neoantigen levels and weak inflammatory signatures.
These patients respond poorly to existing interventions.
In melanoma, 30-40% of patients exhibit innate or acquired resistance.
Innate resistance: initial non-responders.
Acquired resistance: relapse after initial response.
Cold cancers include pancreatic cancer (no response to checkpoint blockade) and prostate cancer (infrequent responses).

Hypoxia and Resistance to PD-1 Blockade

Hypoxia is associated with a lack of response to PD-1 blockade.
Roger Lowe identified a hypoxia-enhanced gene set signature in innate resistant melanoma patients.
Mouse models of prostate cancer show prominent hypoxia, stained with pimonidazole.
Tumor hypoxia predicts poor response to various cancer therapies.
The immune coldest cancers (pancreatic, prostate) have the greatest depth of hypoxia.

T Cell Infiltration and Hypoxic Areas

In normoxic areas of tumors, adequate T cell infiltration is observed.
FOXP3 positive Tregs control the antitumor activity of effector cells.
Hypoxic areas show a lack of T cells.
Without T cells, modern immunotherapy approaches are ineffective.
Similar findings observed in lung cancer models.

Metabolic Environment of Tumors and T Cell Function

The metabolic environment of tumors can be adverse to T cell function, survival, and proliferation.
Hypoxia is linked to multimodal mechanisms of active suppression and nutrient deprivation.
Hypoxia is involved in the differentiation of suppressive phenotypes of MDSC and M2 macrophages, which deplete arginine, tryptophan, and cysteine.
T cells are sensitive to acidic environments and stop proliferating below pH 6.7 and die below pH 6.5.
Lactic acid from the Warburg effect, linked to hypoxia, contributes to a drop in pH.
Hypoxia drives upregulation of carbonic anhydrase IX, creating carbonic acid.
Adenosine release, also linked to hypoxia, binds to A2A and A2B receptors and is broadly immunosuppressive.
Overall, the tumor microenvironment presents a significant challenge to T cell function.

Metabolic Blocks vs. Immune Checkpoint Blockade

Immune checkpoint blockade involves one-to-one suppressive interactions (PD-1, CTLA-4, LAG-3).
Metabolic blocks are more absolute; T cells cannot function in a pH of 6.3 regardless of their numbers.

Mechanisms of Tumor Hypoxia Suppressing T Cell Function

Tumor hypoxia suppresses T cell function through direct and indirect mechanisms.
Indirect mechanisms include conditioning of the vasculature to be cytotoxic to T cells and not favoring adhesion, rolling, and extravasation.
Also, polarization of immature myeloid cells into suppressor phenotypes and upregulation of adenosine-producing enzymes.

Th302 (Evofosfamide): A Hypoxia-Activated Prodrug

Th302 reduces hypoxia in solid tumors, which was discovered serendipitously.
In Tramp-C2 tumors, hypoxia is prevalent (35-40% of the volume).
Checkpoint blockade alone does little to relieve hypoxia.
Th302, alone or with checkpoint blockade, effectively reduces hypoxia.
In KPC-derived pancreatic cancer organoids, Th302 attenuates hypoxia over time.

Mechanism of Action of Th302

Th302 initiates a tissue remodeling process in hypoxic areas.
It kills cells driving abnormal angiogenesis, which are replaced by tissue macrophages (M0 phenotype).
M0 macrophages lay down new, healthy, thick vessels.
These vessels support better T cell extravasation and infiltration, reoxygenation, and breakdown of hypoxia.

Efficacy of Th302 in Mouse Models

Combination checkpoint blockade is effective against Tramp-C2 tumors but more so with Th302.
Th302 alone shows curative efficacy dependent on adaptive immunity, which is lost in Rag knockouts.
Ifosfamide alone cannot replicate the effect; Th302 requires ablation of hypoxic areas for its unique effect.

T Cell Infiltration with Th302 Treatment

Untreated Tramp-C2 tumors have some T cells in normoxic zones but none in hypoxic zones.
Th302 alone increases T cell infiltration; T cells extravasate from thicker vessels.
Checkpoint blockade increases T cell density in normoxic areas but not in hypoxic zones.
The combination of Th302 and checkpoint blockade results in a flood of T cells into normoxic and hypoxic areas.

Reduction of Myeloid Suppressors

Myeloid suppressors thrive in hypoxia; HIF-1 and HIF-2 activation favor MDSC differentiation and acquisition of suppressive features (arginase).
Th302 diminishes CD11b+GR1+ cells, particularly the GR1 subset.
The myeloid compartment is taken over by M0-type macrophages.
The combination therapy reduces the whole tumor frequency of MDSCs by about half.
Arginase-positive CD11b+GR1+ cells are reduced by about 50%.

Functional Impact on MDSCs

The hypoxia that remains in the tumor with treatment does not support MDSC proliferation.
MDSCs from treated animals are less suppressive.
The combination of immunotherapy and Th302 results in a prolonged effect in the tumor microenvironment.

Conditioning of the Tumor Microenvironment

Large tumors were established and treated for one week with Th302 and antibodies, followed by adoptive transfer of congenitally marked bone marrow progenitors.
The combination treatment impairs the ability to recruit and polarize bone marrow myeloid progenitors into effective granulocytic MDSCs.
Absence of immunotherapy in large tumors allows reestablishment of hypoxia.
Maintenance of hypoxia reduction by Th302 requires checkpoint blockade.
Combination treatment affects the suppressive phenotype, so MDSCs phenotype, they fail to upregulate arginase leading to an incomplete development, their suppressive phenotype.

Efficacy in Spontaneous Tumor Models

Spontaneous TRAMP mice develop carcinoma in situ and local metastatic disease by 16 weeks of age.
Treatment started at 16 weeks with Th302 and antibodies for a month, followed by observation until 36 weeks.
Untreated mice have tremendous prostate burden (adenocarcinomas and neuroendocrine prostate cancer).
Checkpoint blockade alone does nothing; tumors resemble untreated animals.
The combination treatment results in normal prostates in about a third of the mice at 36 weeks of age.

Impact on Neuroendocrine Tumors

TRAMP mice develop adenocarcinomas (80%) and neuroendocrine prostate cancer (20%).
The combination group never developed neuroendocrine tumors, which is statistically striking.
The change in the metabolic environment may prevent neuroendocrine development or effectively treat these tumors.

P10 SMAD4 Knockout Prostate Model

Significant extension of survival observed in Rhonda Pino's P10 SMAD4 knockout prostate model (pure adenocarcinomas).
Genetic mutations lead to fewer passenger mutations.

Changes in the Immune Environment

CD8 to Treg ratios improve with the combination of Th302 and checkpoint blockade.
A large amount of granulocytic MDSC exist, with five MDSCs for every one CD8 T cell at baseline.
The combination therapy reverses this, resulting in five CD8s for every one MDSC.
CD4 effector proliferation, CD8 proliferation, and granzyme B production improve with combination therapy.

Antigen-Specific T Cells

High expression of 4-1BB enriches for antigen-specific T cells.
These cells show a higher activation state, higher interferon gamma production, and higher TNF-alpha production.
The metabolic conditioning leads to improved T cell effector function.

Durability of Reconditioning

The reconditioning effect persists over a long time (four months after treatment).
Combination-treated mice maintain better CD8 to Treg and CD4 effector to Treg ratios.
There is a trend toward better CD8 to granulocytic MDSC ratios.

Importance of T Cell Survival

T cells must survive to mediate tumor rejection.
Reactive CD8s undergo apoptosis in the tumor.
If T cells live longer in the tumor, the tumors go away.
The combination promotes extended T cell survival.
In hypoxic areas, actively dying T cells were detected. But with said therapy the rate of death was substantially less.

Clinical Trial at MD Anderson

Advanced cold tumor patients who failed other treatments were treated with evofosfamide and ipilimumab.
18% objective response rate and almost 80% disease control rate were seen.
Responses were seen, including metastatic pancreatic patients with stable tumors for over a year.
The trial was conservative, with two cycles of EVO upfront and standard three mgs per kg Ipi.

Retreatment Results

Patients who had responded well initially were retreated, and nearly all responded upon retreatment.
Better than expected, and the responses that were seen that showed a true response by the subjects being treated.
When patients show enhanced responses upon retreatment, it highlights the importance of the therapy being administered.

Phase Two Trial Design

Longer duration of therapy and added PD-1.
Small patient number but very advanced patients showing promise.
Responses were profound and quite durable.
Advanced prostate cancer patient had about 80% tumor shrinkage, with bone met. It's not clear if it's actually there or not, but his state is in a very good state.

Biomarker Analysis

Small trial, but collected PBMC and did see some results statistically significant.
Statistically significant increases of CD8 T cells in the PBMC.
CD4 T cells also significantly increased in the responders versus nonresponders.
Patients saw higher arginase expression tended to do more poorly.

Biomarkers for Hypoxia

Assess hypoxia reduction, which looks very promising.
Compared signatures via HIF-1 and didn't change/went up versus the responders versus pretreatment.
Hypoxia signature went down in the responding patients versus the untreated patients.
Genomically responders, therapy seems to reduce the hypoxia, and thus correlates with becoming a responder.

Genetic Signatures

Genes that were not responding didn't show in high concentrations of T cells, as the ones that were responding did.
Responders had a lot of preexisting innate inflammation. A very strong genetic signal was made to delineate the difference.
Responders saw increase in innate inflammation. And could make it over the hump with new tumor regression.

Resistance

High oxphos, glycolysis fatty acid, being hypermetabolic and adaptive immune remained disadvantaged with hypoxia's reduction.

Phase 2 Analysis Protocol

Adding PD-1, Evofosfamide, which has show promising results.
Looking at tumor from PET scans to analyze hypothesis.

Additional Oxphos Inhibitor Trial

Looking at HPV vaccine in head and neck cancer for PD-1 to get good outcomes.
But resistance comes high OXPHOS.

Hypoxia and Origin Points

Consumption via abnormal, tumor angiogenesis, which lead to leaky vasculature and disrupted of O2.
As the tumor's upregulate and eat up their resources due to high oxygen consumption, hypoxia tends to rise exponentially.

Studies on Tumor Cells vs Oxygen Metabolism

Studied B16 with CTLA, that wasn't affected.
They were expected to see gene's and protein up and the process to regulate for tumor factors.
CD8 and CD4 expression decrease with decrease in microenvironment activity.
And the microenvironment was able to adapt to the stress given hypoxia

Dealing with OXPHOS Concerns

IAACS developed an inhibitor for cancer cells, which show a preference in tumors.
Combination of all three showed an enhanced and great control over the tumors.
Had to let the tumor establish longer due to the drug being too overactive.
There was proliferation and toxicity mostly CD4 proliferation.
Arginase was lost and tumor was upregulated and it was helping drive it.

Closing and Thank You

Thanks again to Priya Jayaprakash for doing most of the work to make the presentation.
And those with funding being involved to the project as well. Thank you.
The research has been very promising overall in what we are trying to show.