Renata Schmieder Pivetta

Treatment for renal cell carcinoma (RCC) has improved dramatically over the last decade, shifting from high-dose cytokine therapy in combination with surgical resection of tumors to targeted therapy, immunotherapy, and combination therapies. However, curative treatment, particularly for advanced-stage disease, remains rare. Cell therapy as a "living drug" has achieved hematological malignancy cures with a high response rate, and significant research efforts have been made to facilitate its translation to solid tumors. Herein, we overview the cellular therapies for RCC focusing on allogeneic hematopoietic stem cell transplantation, T cell receptor gene-modified T cells, chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) cells, lymphokine-activated killer (LAK) cells, γδ T cells, and dendritic cell vaccination. We have also included perspectives for using other recent approaches, such as CAR macrophages, dendritic cell-cytokine induced killer cells and regulatory CAR-T cells to shed light on preclinical development of cell therapy and advancing cell therapy into clinic to achieve cures for RCC.

• CAIX-targeting immunotherapies decrease PD-L1 expression in ccRCC cell lines. • PI3K/Akt signaling pathways are involved in PD-L1 decrease after CAIX immunotherapy. • CAIX CAR T cell therapy induces immune checkpoint blockade in ccRCC in vivo. The carbonic anhydrase IX (CAIX) expression occurs in most cases of clear cell renal cell carcinoma (ccRCC). This tumor type is characterized by an immunosuppressive microenvironment, where approximately one-fourth of patients overexpress the programmed cell death ligand-1 (PD-L1), significantly increasing their risk of death. Herein, we present a secondary effect of CAIX inhibition using monoclonal antibodies (mAbs) and CAR T cells, leading to PD-L1 downregulation in ccRCC and in vivo immune checkpoint blockade . We identified a positive correlation between CAIX and PD-L1 expression in ccRCC cell lines using in silico RNA-seq data analysis, prompting us to perform fluorescence-activated cell sorting of SKRC52 ccRCC cell subpopulations based on their positive or negative expression of CAIX and PD-L1. After two weeks in culture, the cell population selected to be negative for CAIX and positive for PD-L1 became negative for CAIX and PD-L1. To explore the phenomenon, CAIX blockade was performed using two anti-CAIX monoclonal antibodies (mAbs) in multiple doses in two CAIX+ PD-L1+ clear cell renal cell carcinoma (ccRCC) cell lines. The expression of CAIX and PD-L1 decreased after treatment with the mAbs, and the PI3K/Akt signaling pathway is involved in this modulation. CAIX-targeted CAR T cells in vivo also reduced PD-L1 expression, resulting in superior CD3 infiltration and granzyme B expression, which decreased T cell exhaustion. Our findings demonstrate that CAIX-targeted immunotherapies can induce an indirect immune checkpoint blockade by PD-L1 downregulation. Renal Cell Cancer (RCC) Cells Interacting with T Cells Before and After Anti-Carbonic Anhydrase IX (CAIX) Immunotherapies. A. In the untreated state, RCC cells expressing CAIX and programmed cell death ligand-1 (PD-L1) activate immune checkpoints on T cells, leading to increased expression of PD-1, TIM-3, CD39, and LAG-3. This process results in decreased T cell effector functions, supporting tumor progression. B. After treatment with anti-CAIX antibodies or CAR T cells, RCC cells exhibit reduced expression of CAIX and PD-L1. This action results in immune checkpoint blockade in T cells, characterized by reduced expression of T cell exhaustion markers PD-1, TIM-3, CD39, and LAG-3 and improved T cell effector functions, thereby mitigating tumor development.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most prevalent of renal cancers, with a 5-year survival rate of less than 10% for metastatic cases. The most efficient current strategies to treat ccRCC in advanced settings slightly increase progression-free survival. Chimeric antigen receptor T cells (CAR T cells) targeting carbonic anhydrase IX (CAIX) have reemerged as a promising alternative to ccRCC treatment based on recent preclinical data. CAIX and cyclooxygenase- 2 (COX-2) are key players in tumor progression across various malignancies, overexpressed in 95% and 50% of ccRCC cases, respectively. METHODS: This study employed in silico analysis to examine the expression of CAIX and COX-2 in ccRCC cell lines. The effects of celecoxib, anti-CAIX monoclonal antibodies, and anti-CAIX CAR T cells were evaluated using immunofluorescence microscopy and flow cytometry techniques. RESULTS: Herein, we show a positive correlation between CAIX and COX-2 expression in ccRCC cell lines in vitro and in silico. Notably, COX-2 blockade with celecoxib led to a significant downregulation of CAIX expression in ccRCC cell lines. This effect is retroactive since treatment of these ccRCC cells with two different anti-CAIX monoclonal antibodies (mAbs) resulted in the downregulation of COX-2 expression. The association of celecoxib with anti-CAIX CAR T cell therapy impaired their cytotoxic potential over ccRCC in vitro, depending on CAIX cellular density. CONCLUSION: These findings suggest a regulatory interaction between CAIX and COX-2 levels, indicating that COX-2 inhibitors may diminish the efficacy of CAIX-targeted therapies and should be avoided in combination treatments.

Os autores externos submeteram sua publicação para apresentação de trabalho no evento “International Symposium on Immunobiologicals”, que foi coordenado e organizado pelo Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos), da Fundação Oswaldo Cruz.

Os autores externos submeteram sua publicação para apresentação de trabalho no evento “International Symposium on Immunobiologicals”, que foi coordenado e organizado pelo Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos), da Fundação Oswaldo Cruz.