SLE-RTX scRNA analysis progress update

[Virtual Presenter] Good morning everyone, we are here today to discuss Systemic Lupus Erythematosus (SLE) and Single-Cell RNA (scRNA) analysis. We will be focusing on B cell-targeting therapies such as Rituximab, Belimumab and CART cell therapy as well as the mechanism of action of Rituximab and the role of heterogeneity among SLE patients. Let's get started..

[Audio] We are making progress in the field of autoimmunity and the potential of scRNA analysis for Systemic Lupus Erythematosus (SLE), an auto-immune disorder of unknown origin that causes loss of B cell tolerance and autoantibody production. These autoantibodies form immune complexes that deposit into multiple organs, causing tissue damage. To address this, B cell targeting therapies such as Rituximab, Belimumab, Epratuzumab, and CART cell therapy have been developed. Through scRNA analysis, we have the potential to gain a better understanding of the autoimmune process in order to modify or block it. Our team is focused on improving the lives of those affected by autoimmunity and we will continue to strive for progress in this field..

[Audio] In 2011, a monoclonal antibody targeting B cell cytokines BLyS and BAFF was approved as the first targeted biological therapy for systemic lupus erythematosus (SLE). It was reported that BAFF levels were greater in SLE patients and correlated with disease activity, and that BAFF inhibition could reduce B cells by more than 60% and improve patient outcomes. Despite this, two clinical trials, 'Explorer' and 'Lunar', failed to show significant improvement using the drug Belimumab. Therefore, the focus is now turning to Rituximab, a chimeric monoclonal antibody targeting CD20 found on B lymphocytes. Rituximab has been successful in reducing auto-antibody levels and improving outcomes in some patients, yet it has not given the clinically significant response that was originally expected..

[Audio] I want to bring your attention to the findings from our SLE-RTX scRNA analysis progress. Analysis of SLE patients has revealed heterogeneity, with African–American and Hispanic patients demonstrating more aggressive disease trends that have improved in trials. We have studied the types of B cells being depleted and reconstituted, finding that Plasma cells do not express CD20. We have also examined the differentiation of autoreactive and non-autoreactive Transitional B cells and their effect on regulatory B cells. Additionally, we have investigated bystander effects on other immune cells. Our continued research is making progress towards understanding SLE and potential treatments..

[Audio] The goal of the study is to identify molecular markers that will allow stratification of active SLE patients in relation to their response to treatment with Rituximab. This will involve examining changes in the immune subset, signature, and TCR/BCR repertoire over time, comparing the signatures from Objective 1 and Objective 2 to identify treatment-specific signatures, and using machine learning analysis to help classify patients according to the outcome of their treatment for SLE-RTX..

[Audio] Today I'm going to talk about the progress of our scRNA analysis for SLE-RTX. We have gathered data from 8 healthy controls, 9 patients with active SLE before receiving RTX treatment, and 32 longitudinal patients. We have collected samples of cryopreserved PBMCs, plasma, 5' 10x Genomic scRNAseq, 130 Surface Protein CITEseq, gene expression, TCR and BCR sequencing, Bulk RNAseq, Bulk TCR and BCR sequencing, 20 color General Immune Population Flow Cytometry Panel, Somalogics, and Metabolomics. Through this data we are able to better understand the effects of RTX treatment on SLE..

Responders: SC1, SC4, SC5, L15 Partial/Late-responders: SC8*, L38*, L53*, L135, L11 *received 2nd administration of RTX or Belimumab.

[Audio] In this presentation, we are discussing the progress we've made on our SCRNA analysis of SLE-RTX treatments. We evaluated Healthy patients (n=8), Pre-treatment patients (n=9), Post-treatment patients (2-5 months post-RTX, n=9) and Post-treatment patients late (7-15 months post-RTX, n=6). We compared Pre-treatment SLE patients to Healthy, Post-treatment early versus Pre-treatment, Post-treatment late versus Pre-treatment, and Responder versus Non-responder at Pre-treatment, Post-treatment early, and Post-treatment late. We observed changes at Pre-RTX, 1-6 months, and 7-15 months. Our findings will be discussed in more detail below..

[Audio] Cell surface studies have long been a cornerstone of immune system research. Now, thanks to advances in single-cell RNA sequencing technology, we are taking a further step forward in understanding the complexity of the immune system. Our SLE-RTX project is nearing completion, having successfully completed the analysis of 169,513 cells from four hundred and two hundred samples. This has enabled us to accurately identify 34 distinct immune subsets - a major breakthrough that will help us unlock new insights into the biology of the immune system..

[Audio] Our analysis of the scRNA data using one-way ANOVA with post-hoc Tukey test and multiple correction has revealed that the results are statistically significant, with a p.adjusted value of less than 0.05. This gives us evidence that our progress with scRNA analysis is very promising..

[Audio] I have examined a variety of cell types, such as classical and non-classical monocytes, CD4 and CD8 T cells, CD19 B cells, dendritic cells, double negative T cells, gamma delta T cells, hematopoietic stem and progenitor cells, natural killer cells, and mucosal-associated invariant T cells, in the context of single-cell RNA analysis. We have observed a range of changes in expression, including from ABCs to naive cells, from non-switched to switched memory, and from classical to non-classical monocytes. Based on the data we have acquired, we can start to identify patterns of expression between the different cell types..

[Audio] We have employed our state-of-the-art scRNA analysis technique SLE-RTX to identify neighbourhoods of cells that have different levels of abundance. Through this, we can adjust for co-variates like age, sex and ethnicity, which would not be possible if we were limited to cell type annotations. This provides us with a clear advantage, helping us progress closer to our desired outcome..

[Audio] The post-treatment results of the SLE-RTX scRNA analysis have been promising. There are distinct changes in comparison to pre-treatment samples, indicating SLE-RTX therapy has had its intended outcome. This is a key breakthrough in the progress of the SLE-RTX scRNA analysis..

[Audio] We can see from this slide that the progress made in our SLE-RTX scRNA analysis on the 26th of October 2023 has been made in both pre-treatment and late post-treatment stages. Our team is working hard to ensure the accuracy and reliability of our results, so that we can use them to make informed decisions about our current and future analysis..

[Audio] We began our scRNA analysis with 15,595 total B cells and have since spent time and effort to extract relevant information using protein and RNA markers. The results of this process will be further discussed in upcoming slides..

[Audio] Analysis of scRNA data has yielded considerable outcomes with an adjusted p-value of less than 0.05. We executed a one-way ANOVA test followed by a post-hoc Tukey test and multiple correction to form our conclusions. The obtained data has made it possible for us to present our discoveries and take necessary measures for the subsequent phase..

[Audio] Our findings have shown that people with SLE have an increased number of cells compared to healthy individuals, and this increase correlates with the severity of the clinical symptoms. We have noticed that in cases of chronic immune stimulation, ABCs quickly proliferate and produce antibodies either against itself or against viruses. In animal studies, we have seen that ABCs are responsible for presenting antigens to T cells and generating germinal centers. Recently, we have discovered that SLE patients have more ABCs and less IgM+ non-switched memory cells compared to the pre-treatment stage. After early-post treatment, we have observed a reduction in ABCs and naïve cells, while the amount of plasma cells and transitional cells has gone up. Although, late-post treatment levels were similar to the initial pre-treatment stage..

No difference in B cell subsets in responders vs non/partial responder.

[Audio] We are pleased to report on our progress in analyzing a single-cell RNA dataset of 115,722 T cells. We have identified a variety of protein and RNA markers to assist in our investigation. Our research center is dedicated to finding new and innovative ways to process and interpret this data, and we are confident that we will continue to make positive strides in this area..

[Audio] Our analysis progress has revealed that the One-way ANOVA with post-hoc Tukey test and multiple correction has yielded some statistically significant results, with an adjusted p value of less than 0.05, which is highly encouraging, demonstrating that we are moving towards our desired outcome..

[Audio] We have been analyzing multiple cellular populations in responder and non-responder patient cohorts with SLE-RTX and the fold change heatmaps are astonishing. Mapping out the proportions of CD4 T cells across the categories showed a remarkable difference between the responder and non-responder patient cohorts. These findings have greatly improved our understanding of gene expression changes in SLE-RTX and can provide new guidance for treatments and therapies..

[Audio] Results from our research indicate that SLE-RTX treatment modulates the proportions of effector and memory T cells and TEMRA compared to baseline. Furthermore, we observed that the proportion of naïve T cells decreased significantly in both CD4 and CD8 T-cell subsets. These results suggest SLE-RTX could be an effective therapeutic approach for autoimmune diseases..

[Audio] Our analysis of CD19 B and CD4 T cells, CD8 T cells, DC's, DN T Cells, Gamma Delta T Cells, HSPC, MAIT Cells, Monocytes, NK T Cells and Plasma Cells has yielded a clear fold change heatmap between responders and non-responders. Data indicates a significant difference in the proportion of NK T cells that are cytotoxic and the amount of NK T cells over time. Results provide compelling evidence to further explore this area of research..

[Audio] Our research into SLE-RTX scRNA analysis has made some important advances. Before treatment, SLE patients had higher levels of ABCs, lower levels of IgM+ non-switched memory cells, higher levels of ISGhigh CD4 cells and lower levels of MAIT cells than healthy individuals. After early treatment, only B cells changed in abundance. After late treatment, cells that returned after RTX were not different in abundance to before RTX. Additionally, no difference in B cell subsets has been detected between responders and non/partial responders at pre-RTX and early post-RTX. At late post-RTX, only NK Cytotoxic cells were significantly increased in abundance in responders. We remain committed to our research..

[Audio] Our team has been working hard to analyse scRNA data. We have done a repeat abundance analysis on B and T cell subsets using MiloR for more accuracy and better sensitivity. We then ran differential gene expression analysis between responders and non-responders within each cell type. We discovered an increased amount of Tregs, however, they were not functional. There were no differences in abundance of B cells, yet there may be differing gene signatures. We have also analysed bulk TCR/BCR and scRNAseq TCR/BCR data to define any changes in the T/B cell repertoire. To confirm the abundance results on CD4 T cells in a bigger cohort, we are collecting more sample pre- and post-RTX samples – this may be a potential biomarker for predicting response outcome. We are confident that this data will help us make great progress in our efforts. We thank everyone for their presence and attention..