Our Research

Dr. Ruella's laboratory focuses on the study of the mechanisms of relapse after chimeric antigen receptor T cell (CART) immunotherapy with the goal of rationally design innovative next-generation immunotherapies for relapsing/refractory leukemia and lymphoma. We use the most recent technologies such as CRISPR-Cas9, single-cell RNA-sequencing, digital spatial profiling, imaging mass cytometry, and others to understand the intricate interactions between immune cells, cancer cells, and the tumor microenvironment. We are using cutting-edge technologies (CRISPR-Cas9, functional genomics, single-cell RNA-seq., imaging mass cytometry, digital spatial profiling etc.) to advance innovative research ideas inspired by the unmet needs that we observe in the clinic. We are particularly interested in the mechanisms of relapse after CART immunotherapy and the development of innovative therapeutic strategies to overcome resistance. We bring new ideas from the laboratory to clinical trials thanks to the supportive environment of the University of Pennsylvania.

Several projects are currently being explored in the laboratory, including:

1. Enhancement of CART activity with small molecules. Several novel small molecules are or will be soon approved for leukemia and lymphoma. Despite initially high response rates, durable remissions are not common. The goal of this project is to increase the durable disease control in hematological cancers by combining novel small molecules with CART. We previously demonstrated the potent activity of the combination of ibrutinib (BTK inhibitor) and CART-19 for Mantle Cell Lymphoma. Moreover, we showed for the first time that ibrutinib can modulate CAR T cell functions (PMID: 26819453 and Patent US20150283178A1 and US2016164580A1). This work was recently translated into a successful clinical trial (Gill S. ASH 2018 #298). We are now studying pro-apoptotic drugs such as SMAC mimetics that synergistically increase CART killing (Singh N. Cancer Discov. 2020 Jan 30)

2. Mechanisms of resistance to CART immunotherapy. Despite the description of several mechanisms of relapse after CART19, several cases remain unexplained. We previously reported a patient relapsing after CART19 with CD19-neg leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T-cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019 (PMID: 30275568). Another approach we took to understand resistance mechanisms is functional genomics. Using a genome-wide loss-of-function screen, we identified the death receptor pathway as a central regulator of primary CART19 resistance. We found that impaired death receptor signaling in ALL leads to rapidly progressive disease despite CART19 treatment, mediated by an initial inherent resistance to T cell cytotoxicity that was subsequently magnified by the induction of CAR T cell dysfunction. This observation was extended to clinical samples from two distinct CART19 trials in ALL, where we found that reduced expression of death receptor genes was associated with primary CART19 failure, reduced CAR T cell fitness, and worse overall survival (Singh N. Cancer Discov. 2020 Jan 30). Lastly, to define the critical predictors of response to CART19 in lymphoma, we established a collaborative platform to collect peripheral blood samples and serial tumor biopsies from patients receiving CART and study them using the most advanced genomic, epigenomic and proteomics technologies.

3. Targeting CD19-negative relapses occurring after CD19-targeted immunotherapies. Potent CD19-targeted immunotherapies (are generating impressive results in the setting of B-cell acute lymphoblastic leukemia (B-ALL) and lymphoma. However, a significant subset of patients still relapses, and the majority of relapses are characterized by loss of detectable CD19. This phenomenon has been particularly relevant in B-ALL, but it is also observed in DLBCL. We previously demonstrated that targeting CD123 together with CD19 can reduce antigen-loss escape in preclinical models (Patent US2016/028896A1). We showed that T cells that can express two CAR (dual CART) (CART19/123) are more effective than pooled CART (CART19+CART123) (PMID: 27571406). We are now developing additional dual CARTs targeting CD19 and CD22 (and other targets) (Patent US20160362472A1 and Ruella M. Oral presentation ASH 2017; manuscript under revision).

4. Chimeric Antigen Receptor T cells to target the lymphoma microenvironment. In Hodgkin lymphoma (HL) about 95% of the tumor mass consists in the reactive tumor environment, including tumor-associated macrophages (TAM). At the same time, only a small subset of the total cells represents the neoplastic Reed-Sternberg cells (HRS). We demonstrated that CAR T cells against CD123 can kill in vitro and in vivo HL cells and can also target CD123+ TAMs, which lead in return to better CART function. (Ruella Cancer Discovery 2017 and Patent WO2018026819A3). We are now expanding our observations to other lymphomas, using single-cell RNA sequencing, and developing strategies to target TAM.

5. Cytokine-release syndrome after CART therapies. CART19 generate impressive responses in B-cell leukemia and lymphoma. However, cytokine-release syndrome (CRS), a severe systemic inflammation due to a massive release of cytokines by activated CAR T cells and other immune cells, can lead to serious toxicities including death, therefore limiting the use of this promising immunotherapy in many patients. The goal of this project is to generate a clinically-relevant model of CRS and studying possible strategies to reduce and prevent CRS. Our results show that ibrutinib can reduce CRS in xenografts models of MCL treated with CART (PMID: 27677739 and patent WO2018013918A3).