The effect of anti-PD-L1 atezolizumab and nanobody Nb202 immunotherapy on glioblastoma patient-derived organoids

Glioblastoma is the most common malignant brain tumor and one of the deadliest cancers. Despite receiving standard therapy which includes maximal safe surgical removal of the tumor, radiotherapy and chemotherapy, the majority of patients succumb to the disease within 2 years. Due to low therapy success, new and more efficient drugs and treatment strategies are urgently needed. One of the treatment options that proved to be exceptionally efficient in some cancers is immunotherapy with immune checkpoint inhibitors, especially inhibitors of PD-1/PD-L1 axis. Although up to now, glioblastoma has shown high resistance to anti-PD-1 and anti-PD-L1 inhibitors, in vitro results implicate that glioblastoma may respond to them in similar manner as other tumor. Also, results of clinical trials show that a subset of patients respond to therapy, which indicates that there is a large room for improvement for immunotherapy in glioblastoma. One of such improvements are use of nanobodies. Nanobodies are antigen-recognizing parts of heavy-chain only antibodies produced by just few animals, for example llamas and sharks. They have unique and advantageous characteristics, e.g., better tumor penetration compared to classical antibodies, high stability, high affinity, and non-immunogenicity. In my project I plan to validate the cytotoxic effect of anti-PD-L1 nanobody (Nb202), that has been already generated in our lab. We have previously shown with surface plasmon resonance that Nb202 is specific to PD-L1 and has high affinity (Kd=0.644 µM; preliminary and unpublished results). We have also shown that the expression of PD-L1 is more than 50x higher in glioblastoma tissue compared to reference tissue which makes glioblastoma a suitable target for PD-L1 inhibitors. For the efficacy evaluation purpose, we will first generate glioblastoma patient-derived organoids, that would retain components of immune system to enable efficient immunotherapy testing. For organoid plating, I will use air-liquid interface system, which has been confirmed as especially efficient system for organoid culture containing original microenvironment, including immune system. First, organoids will be prepared from glioblastoma tissue. After organoid establishment, we will perform RNAseq on samples from original tissue and organoids. From gene expression we will generate heatmap for each tissue-organoid pair and estimate how well do organoids reflect original tumor. We will also determine the expression of PD-1 and PD-L1 by western blot as well as the presence of specific immune cells using immunohistochemistry. Organoids will then be treated with monoclonal antibody atezolizumab and nanobody Nb202, both inhibitors of PD-L1, using the same concentrations that were observed in serum of patients when treated with atezolizumab. Decrease of growth will be determined by organoid diameter change as well as with WST-1 cytotoxic test. Using histochemistry, we will also analyze the location of atezolizumab and Nb202, as we predict, that due to its low size Nb202 penetrates organoid better compared to classical antibody, atezolizumab. At last, expression of PD-L1 and PD-1 as well as the presence of immune cells will be analyzed in treated organoids. Therefore, the purpose of the proposed study is to evaluate potential use of Nb202 and atezolizumab as immunotherapeutic agents in glioblastoma. This research will bring us a step closer to the understanding of glioblastoma tumor response on immunotherapy, especially in context of different treatments modalities. Moreover, development of patient-derived organoids could in future enable more precise and reliable drug testing. Organoids could eventually predict patients’ response to drugs more accurately, taken into consideration the biology of the tumor and its microenvironment as a whole.