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Tackling the Achilles Heel of Immunotherapy: Validating imaging biomarkers and targeting the immunological niche of tumour hypoxia

Periodic Reporting for period 3 - HYPOXIMMUNO (Tackling the Achilles Heel of Immunotherapy: Validating imaging biomarkers and targeting the immunological niche of tumour hypoxia)

Reporting period: 2019-12-01 to 2021-05-31

The absence of effective anticancer treatment, in combination with the steeply rising prevalence of cancer, underscores the urgent need for more specific and effective cancer therapies in combination with biomarkers to select the right patient for the right treatment. Overall, this is a typical project of convergence sciences sitting at the intersection of clinical sciences (WP4), biology (WP1-2) and technology (WP3).

Immune checkpoint inhibitors (ICIs), monoclonal antibodies relieving T-cells from their negative regulation, elicit robust anti-tumour immune responses in advance-staged cancer patients, however with only limited durable responses. Accumulating evidence suggests that oxygen deficiency (hypoxia) within tumours is an important phenomenon to suppress the anti-tumour immune response. Therefore, we hypothesised that targeting hypoxia may improve the efficacy of immunotherapy through lowering the tumour hypoxic fraction, using a next generation hypoxia-activated prodrug (HAPng). Only under severe hypoxic conditions, the HAPng leads to the generation of active metabolites to exert its cytotoxic effect as DNA crosslinker ( We hypothesized a beneficial therapeutic outcome of a trimodal therapy, more specifically the combination of immunotherapy (“pushing the accelerator” with L19-IL2 and “releasing the break” with checkpoint inhibitor ( with HAPng. We have obtained experimental evidence that HAPng can increase the immunological visibility of tumours via an increase in immunogenic cell death (ICD). In addition, using the concept of HAPs, we will bring immunomodulators targeting TLR7/8 in these hypoxic areas in order to stimulate the activation of pro-inflammatory cytokines.

Another way to tackle hypoxic cells is to use radiotherapy with high linear energy transfer (LET) such as carbon ions. Compared to conventional photon radiotherapy, carbon ions will have a higher deposition of the actual radiation dose at the tumour site without being influenced by hypoxia. Experiments have been completed in collaboration with DKFZ (Heidelberg).

Furthermore, to establish an ideal individualized therapy, the identification of biomarkers of therapy efficacy is similarly important. Thus, it would be beneficial for patients as well as being cost-effective if the outcome of therapy could be predicted based on biomarker(s) assays to guide the most optimal treatment selection for each individual patient. We envision biomarker(s), preferably tri-dimensional, imaging-based and non-invasive such as radiomics approaches, as a powerful tool to maximize a therapeutic benefit for patients.
We have demonstrated that the synergistic effect of radiation is greater with immunocytokine then with checkpoint inhibitor and that combined immunotherapy is more efficient then one of the two single approaches.

The HAPng has been investigated thoroughly as monotherapy in several tumour models showing a therapeutic efficacy independent of the baseline hypoxic fraction. Using knockout cell lines we were able to show that HAPng activity depends on Homologous Recombination (HR) and Fanconi Anemia (FA) status. HAPng also is capable of sensitizing tumours to irradiation, immunotherapy (immunocyokines or checkpoint inhibitors) and chemotherapy, see Figure ' Survival Curves'. Based on these data, we will also investigate the approach of bringing immunomodulators in hypoxic areas, by using new “HAP-immunomodulators”. The synthesis of these compounds is currently ongoing.

We tested in collaboration with Heidelberg the combination of Carbon ion irradiation with different LET and immunocytokines. Although high LET irradiation had a therapeutic benefit compared to X-rays, the combination with immunocytokines did not result in increased benefit. Currently, we are investigating the molecular mechanisms in order to explain these results. Clonogenic survival analyses using different cell lines have been performed to calculate the relative biological effectiveness (RBE) of the different LET irradiations, allowing in the future irradiation with RBE adjusted dose in a fractionated manner.

Thanks to numerous advances in the field of radiomics image analysis (Lambin et al., Nat Rev Clin Oncol, 2017), we are making great progress in our ability to implement radiomics imaging biomarkers to improve patient selection. In parallel, there are multiple steps still requiring further rigorous attention and are currently under investigation. We have managed to generate validated hypoxia signatures on both CT and FDG-PET, with a radiomics approach. We found that the use of nitroglycerin (a repurposed vasodilating drug) patching to target hypoxia, did not result in improved outcome. We gathered and curated image data to validate currently published immunotherapy response CT signatures. Worthwhile to mention are key technological achievements such as the use of longitudinal cone beam CT data for radiomics analysis.

After completion of the phase 1 trial (NCT02086721), the Medical Ethical Committee in The Netherlands and Belgium approved the ImmunoSABR phase 2 trial ( Within this trial, we investigate the Progression Free Survival after double or triple therapy compared to SOC in stage IV NSCLC patients (< 10 metastasis). >20 patients have been enrolled in this trial.
A. Pre-clinical work (WP1-2)
- Further characterization of the DNA damage response to HAPng
- Identification of potential biomarkers of response to HAPng
- Testing the combination of HAPng with standard-of-care treatments
- Evidence that removing the immunoresistant hypoxic areas will sensitize tumours to immunotherapy and reveal the molecular mechanism
- Test the therapeutic efficacy of fractionated high LET irradiation (RBE-adjusted dose) in combination with immunotherapy and reveal the molecular mechanism
- Test intratumoural clostridium as delivery system of immunotherapeutics ( a project for which we received a ERC PoC.

B. Radiomics as biomarker (WP2)
- A fully automated lung tumour segmentation algorithm, a project for which we received a second ERC PoC
- A radiomics signature that can
1) detect lung cancer histologic subtype
2) accurately predict tumour oxygenation status
3) accurately evaluate immunotherapy response
4) predict patients likely to develop pneumonitis after immunotherapy.

C. Clinical trials (WP4)
Regarding ImmunoSABR (phase 1 completed, randomized phase 2 running: we will:
- Investigate the combination radiotherapy and immunotherapy alone vs triple therapy (radiotherapy + Immunocytokine + checkpoint inhibitor)
- Use this information for the next trial adding an HAP.
- Use all CT-scans to validate the Hypoxia radiomics signature
- Predict Predicting early death in NSCLC patients
- Predict tumour hypoxia in NSCLC patients based on FDG and HX4 PET scan
- Predict tumour hypoxia based on CT radiomics
- Start a third trial (Phase 1 with HAPng) in 2021.

Overall this project is very successful (> 50 papers, three patents, three clinical trials, two ERC PoC) and is a perfect example of convergence sciences, integrating knowledge form clinical sciences, technology and biology.
Survival Curves