Overcoming microenvironmental resistance to PD-1 blockade in genetically engineered lung cancer models

Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of nonsmall cell lung cancer (NSCLC). However, only a minority of patients respond, and sustained remissions are rare. Both chemotherapy and antiangiogenic drugs may improve the efficacy of ICB in mouse tumor models and patients with cancer. Here, we used genetically engineered mouse models of Kras(G12D/+);p53(-/-) NSCLC, including a mismatch repair-deficient variant (Kras(G12D/+);p53(-/-);Msh2(-/-)) with higher mutational burden, and longitudinal imaging to study tumor response and resistance to combinations of ICB, antiangiogenic therapy, and chemotherapy. Antiangiogenic blockade of vascular endothelial growth factor A and angiopoietin-2 markedly slowed progression of autochthonous lung tumors, but contrary to findings in other cancer types, addition of a PD-1 or PD-L1 antibody was not beneficial and even accelerated progression of a fraction of the tumors. We found that antiangiogenic treatment facilitated tumor infiltration by PD-1(+) regulatory T cells (T-regs), which were more efficiently targeted by the PD-1 antibody than CD8(+) T cells. Both tumor-associated macrophages (TAMs) of monocyte origin, which are colony-stimulating factor 1 receptor (CSF1R) dependent, and TAMs of alveolar origin, which are sensitive to cisplatin, contributed to establish a transforming growth factor-beta-rich tumor microenvironment that supported PD-1(+) T-regs. Dual TAM targeting with a combination of a CSF1R inhibitor and cisplatin abated T-regs, redirected the PD-1 antibody to CD8(+) T cells, and improved the efficacy of antiangiogenic immunotherapy, achieving regression of most tumors.

Anti-angiogenic immunotherapy for lung cancer

Ece Kadioglu

Lung cancer is the leading cause of cancer-associated deaths worldwide. Platinum-based chemotherapy is the most common therapeutic approach in advanced non-small cell lung cancer (NSCLC), particularly for tumors that carry non-druggable activating mutations. However, these tumors are generally not eradicated when diagnosed at an advanced stage and are frequently resistant to chemotherapy. Therefore, there is an urgent medical need for new treatments for this frequent cancer type. Immunotherapies are treatments based on increasing the strength of immune responses against cancer. In particular, monoclonal antibodies that block inhibitory "immune-checkpoints" expressed on T cells (e.g., programmed cell death protein 1, or PD1) show clinical efficacy in increasing tumor types. However, these therapies improve survival in only a minority of NSCLC patients, and ongoing efforts are being made for increasing their efficacy through combinatorial treatments. In this regard, growing evidence indicates that tumor angiogenesis is closely associated with immunosuppression. Pro-angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (ANG2), cause abnormalities and dysfunctions in tumor blood vessels that impair T cell extravasation and anti-tumoral activity. Recent findings in preclinical cancer models show that reprogramming the features of tumor-associated blood vessels by anti-angiogenic drugs may help to increase intratumoral T cell abundance and sensitize refractory tumors to immunotherapy. Nevertheless, the potential of anti-angiogenics in the context of lung cancer immunotherapy is poorly understood. Therefore, the main objective of my project was to audit combinations of anti-angiogenic therapy and immune checkpoint blockade in a genetically engineered mouse model of NSCLC. I first studied the anti-tumoral activity of dual VEGFA and ANG2 inhibition using a bispecific antibody (A2V) in the KrasLSL-G12D/+;p53fl/fl (KP) NSCLC model. I found that A2V, but not single VEGFA or ANG2 blockade, had robust tumor-inhibitory activity in KP mice, which was at least comparable to standard-of-care chemotherapy. However, A2V only moderately increased cytotoxic T cells in the tumors. To potentially increase the immunogenicity of KP tumors, I also generated two KP mouse variants by either co-expressing a surrogate neoantigen or genetically deleting a DNA mismatch repair enzyme in the cancer cells. However, the therapeutic efficacy of A2V was not further improved in spite of a slightly more activated immune response. I next investigated combination therapies involving A2V and PD1 blockade. In contrast to previous findings in other cancer types, the addition of PD1 antibodies deteriorated the efficacy of A2V, likely through enhancing immunosuppressive mechanisms sustained, at least in part, by regulatory T cells and T cell-antagonizing cytokines. In summary, my work supports the potential clinical efficacy of combined VEGFA and ANG2 blockade for lung cancer therapy but provided little evidence for its synergy in association with anti-PD1 therapy.

EPFL2019

Combinatorial targeting of angiogenesis with inducers of autophagy in the treatment of glioblastoma multiforme

Julie Laetitia Scotton

Malignant gliomas represent 80% of tumors developing in the central nervous system, with 50% being glioblastomas (GBM), the most aggressive form of the disease. The benefit from current therapies is very limited, the overall 5-year survival rate of patients with GBM being less than 5%. Thus, there is an urgent need for better therapies. It has been reported that the use of tricyclic antidepressants (TCAs) is linked with reduced incidence of gliomas. In line with these observations, our lab previously demonstrated that combining imipramine (IM), a TCA that activates adenylate cyclase and elevates production of cAMP, with ticlopidine (TIC), an inhibitor of the purinergic receptor P2Y12 that otherwise suppresses cAMP, results in a concerted increase in the levels of intracellular cAMP, which consequently stimulates increased autophagy in glioma cells, leading to autophagy-associated cell death (AACD) in culture and during orthotopic tumor growth. In several mouse models of glioma, the combination improved survival and reduced malignant grade. Seeking to assess the therapeutic translational potential of these repurposed drugs, particularly IM, we performed pre-clinical trials combining IM+/-TIC with anti-VEGF therapy, which is commonly used in patients with GBM; such anti-angiogenic therapy is associated with a transitory improvement in progression-free survival and quality of life, but does not translate into an overall survival benefit. Using genetic mouse models with enabling bioluminescent monitoring of the cancer cells, we treated established GBMs with imipramine, ticlopidine, and B20S, an anti-mouse VEGF monoclonal antibody. We observed transient stabilization of tumor growth, and the overall survival was significantly extended in the regimens combining B20S with IM +/- TIC compared to control and single treatments. Similar to clinical observations in GBM patients treated with anti-angiogenic therapy, mice treated with B20S alone had no survival benefit, implicating a synergetic effect for the combination of anti-VEGF treatment with imipramine and ticlopidine. We found that the combinatorial therapy elicited further increases in autophagy and displayed some indication of blood vessel normalization, concomitant with an elevation of CD8+T lymphocyte infiltration into the orthotopic tumors. Notably, we could partially reverse the beneficial effect of the combinations using a CD8+T cell-depleting antibody, revealing the involvement of the recruited CD8+T cells in the efficacy of the combinatorial treatment, in addition to the cell-intrinsic autophagy-associated death that is demonstrable in culture and in tumors. Together, these results suggest a strategy to improve anti-angiogenic therapy in patients with glioblastoma, by using repurposed FDA-approved drugs that markedly elevate autophagy in concert with anti-VEGF therapy. The recruitment of CD8+ T cells into the treated tumors and their evident contribution to therapeutic efficacy motivates ongoing experiments aimed to heighten and sustain the induced anti-tumor immune response.

EPFL2018

An autonomous microfluidic device for identification and analysis of individual clinically-relevant mammalian cells

Fabien Louis Claude Robert Jammes

Cancer represents one of the major public health challenges worldwide, representing more than 25% of all deaths in the European Union in 2014. It affects a large and growing part of the general population, with the National Cancer Institute (NCI) estimating that nearly 40% of men and women will be diagnosed with cancer at some point in their lifetimes.Cancer presents an enormous intrinsic complexity and diversity as well as a pronounced resistance to interventions. Current treatments are composed primarily of chemotherapy, surgery, radiotherapy and drug therapy. Each of these approaches has been met with restrained successes and limitations. One growing field of cancer treatments that is showing promising results is immunotherapy. An improved understanding of the cancer biology and microenvironment over the years, particularly the interactions between a tumor and the immune system, has demonstrated the capability of the host immune system to detect and eliminate malignant cells through the process of immune surveillance. However, by various mechanisms, malignant cells can stochastically "hide" from the immune system and transform into tumors. Immunotherapies intend to use the immune system to fight the tumor progression. Different kinds of immunotherapies exist: from immune checkpoint inhibitors (PD-1), to monoclonal antibody treatments and cancer vaccines. One interesting approach of immunotherapy is called adoptive cell transfer therapy (ACT), wherein immune cells, and more precisely, T cells, from the cancer-affected patient are extracted, allowing tumor-specific and effective T cells to be isolated, expanded, activated and re-injected into the patient's body. This type of treatment has already demonstrated great tumor clearance potential as well as the ability to induce long-term cancer immunity. However, one of the major challenges in this type of treatment is the recognition, isolation and expansion of high-affinity, effective T cells that are able to eliminate cancer cells. Several key advances in the field of genetics, sequencing, and personalized medicine have allowed for the identification of tumor-specific antigens, the unique cancer signature for a given patient and a specific tumor. But there is a growing need for novel tools and platforms enabling the screening, selection and isolation of rare, naturally-occurring, high-affinity, tumor-specific T cells. In this thesis we present a novel approach to study the affinity of CD8+ lymphocytes at the single-cell level using microfluidics technology. We demonstrate the ability of our platform to isolate single cells and evaluate their affinities towards a given antigen by using the reversible pMHC NTAmers technology developed by Julien Schmidt et al. at the Ludwig Institute for Cancer Research in Lausanne, Switzerland. The innovative platform developed here effectively alleviates some of the bottlenecks of current technologies, like flow cytometry, for affinity studies of lymphocytes at the single-cell level. This thesis represents the development from idea to proof-of-concept of this novel platform and we herein describe the origins and characterization of this technology as well as the first applications towards a personalized-medicine tool for clinical research uses.

EPFL2021