Antibodies against CD20 or B-Cell Receptor Induce Similar Transcription Patterns in Human Lymphoma Cell Lines

BACKGROUND: CD20 is a cell surface protein exclusively expressed on B cells. It is a clinically validated target for Non-Hodgkin's lymphomas (NHL) and autoimmune diseases. The B cell receptor (BCR) plays an important role for development and proliferation of pre-B and B cells. Physical interaction of CD20 with BCR and components of the BCR signaling cascade has been reported but the consequences are not fully understood. METHODOLOGY: In this study we employed antibodies against CD20 and against the BCR to trigger the respective signaling. These antibodies induced very similar expression patterns of up- and down-regulated genes in NHL cell lines indicating that CD20 may play a role in BCR signaling and vice versa. Two of the genes that were rapidly and transiently induced by both stimuli are CCL3 and CCL4. 4 hours after stimulation the concentration of these chemokines in culture medium reaches a maximum. Spleen tyrosine kinase Syk is a cytoplasmic tyrosine kinase and a key component of BCR signaling. Both siRNA mediated silencing of Syk and inhibition by selective small molecule inhibitors impaired CCL3/CCL4 protein induction after treatment with either anti-CD20 or anti-BCR antibodies. CONCLUSION: Our results suggest that treatment with anti-CD20 antibodies triggers at least partially a BCR activation-like response in NHL cell lines

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Determining significant fold differences in gene expression analysis

A typical use for RNA expression microarrays is comparing the measurement of gene expression of two groups. There has not been a study reproducing an entire experiment and modeling the distribution of reproducibility of fold differences. Our goal was to create a model of significance for fold differences, then maximize the number of ESTs above that threshold. Multiple strategies were tested to filter out those ESTs contributing to noise, thus decreasing the requirements of what was needed for significance. We found that even though RNA expression levels appear consistent in duplicate measurements, when entire experiments are duplicated, the calculated fold differences are not as consistent. Thus, it is critically important to repeat as many data points as possible, to ensure that genes and ESTs labeled as significant are truly so. We were successfully able to use duplicated expression measurements to model the duplicated fold differences, and to calculate the levels of fold difference needed to reach significance. This approach can be applied to many other experiments to ascertain significance without a priori assumptions.

Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies

CD20 is a cell-surface marker of normal and malignant B cells. Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by both types. We also determined the first X-ray structure of a type II antibody by crystallizing the obinutuzumab (GA101) Fab fragment alone and in complex with a CD20 cyclopeptide. Despite recognizing an overlapping epitope, GA101 binds CD20 in a completely different orientation than type I antibodies. Moreover, the elbow angle of GA101 is almost 30° wider than in type I antibodies, potentially resulting in different spatial arrangements of 2 CD20 molecules bound to a single GA101 or rituximab molecule. Using protein tomography, different CD20 complexes were found to be associated with the 2 antibodies, and confocal microscopy showed different membrane compartmentalization of these subpopulations of the cellular CD20 pool. Our findings offer a possible molecular explanation for the different cellular responses elicited by type I and II antibodies