Abstract B051: Marrow and peripheral blood cytokine architecture differences between pediatric patients receiving CD19- and CD22-directed CART
Authors: Diorio Caroline , Uppuluri Lahari , Thadi Anusha , Myers Regina , Shraim Rawan , Dinofia Amanda , Martinez Zachary , Elhachimi Amira , Fraietta Joseph , Gonzalez Vanessa , Yi Vivian , Newman Haley , Hughes Andrew , Tan Kai , Grupp Stephan , Teachey David T. 
Published: September 2024

Abstract
Introduction: Chimeric antigen receptor T-cell (CART) targeting CD19 (CART19) have revolutionized therapy for relapsed/refractory pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, not all children are cured with CD19-directed CART, leading to the use of CD22-directed CART (CART22) as an alternative therapy. To better understand differences in outcome and toxicity following CART19 and CART22 we used high-throughput protein and transcriptional techniques to understand differences in cytokine architecture. Methods: We used a proximity extension assay (Olink Signature 100 T48, Olink, Uppsala, Sweden) to measure 48 proteins from paired peripheral blood (PB) and bone marrow (BM) serum from 20 patients (N=10 CART19, N=10 CART22). We performed single cell RNA sequencing (scRNAseq) on pre-infusion peripheral blood mononuclear cells (PBMCs; N=3 CART19, N=4 CART22) and pre-infusion bone marrow mononuclear cells (BMMCs; N=3 CART19, N=4 CART22). Croypreserved cells were thawed. Approximately ten thousand cells from each sample were pooled into a single volume and then taken into a single cell RNA-Seq assay using the Chromium NextGem Single Cell 3' GEM, Library & Gel Bead Kit v3.1(10X Genomics). Sequencing libraries were run on Bioanalyzer 2100 and quantified with KAPA library quantification kit (Roche 07960140001) prior to sequencing on Illumina NovaSeq6000 with 28:10:10:90 paired-end configuration. Results: Samples were obtained from patients treated on clinical trials of CART19 (NCT01626495, NCT02906371) and CART22 (NCT02650414). There was no difference in pre-infusion disease burden between CART19 and CART22 patients. All patients who received CART22 had previously been treated with CART19 or the CD19 directed bispecific T-cell engaging antibody blinatumomab. Pre-treatment marrow serum from patients who went on to receive CART22 had higher levels of the proteins CSF3, IL10, CXCL9, IL6, CXCL8 and CXCL10, implying that previous treatment with CART19 impacted the marrow environment. Pre-treatment marrow cellular populations also differed between CART19 and CART22 patients. Patients who went on to receive CART22 had a higher proportion of CD16+ monocytes then CART19 patients. Three distinct clusters of CD16+ monocytes were identified with unique transcriptional profiles. We previously demonstrated that the anti-inflammatory cytokine IL-10 was higher in the peripheral blood of patients who went on to receive CART22. Measured IL-10 protein levels were higher in both BM and PB serum. Pre-treatment PBMC expression of IL-10 measured by scRNAseq did not differ appreciably between patients who went on to receive CART19 and those who went on to receive CART22. However, IL-10 expression in BMMCs was higher in patients who went on to receive CART22, implying that measured IL-10 differences have a marrow cellular source. Conclusions: Using high throughput protein measurement techniques and scRNAseq we demonstrate clear differences in the pre-infusion cytokine and cellular architecture of patients treated with CART19 or CART22.
Citation Format: Caroline Diorio, Lahari Uppuluri, Anusha Thadi, Regina Myers, Rawan Shraim, Amanda Dinofia, Zachary Martinez, Amira Elhachimi, Joseph Fraietta, Vanessa Gonzalez, Vivian Yi, Haley Newman, Andrew Hughes, Kai Tan, Stephan Grupp, David T. Teachey. Marrow and peripheral blood cytokine architecture differences between pediatric patients receiving CD19- and CD22-directed CART [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B051.