Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness.

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation, which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared with conventional CARs and demonstrated the up-regulated genes associated with T cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B cell lymphoblastic leukemia and B cell non-Hodgkin lymphoma xenograft models while demonstrating persistent T cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

Proteome Analysis of the Antiproliferative Activity of the Novel Chitooligosaccharide-Gallic Acid Conjugate against the SW620 Colon Cancer Cell Line.

Chitooligosaccharide (COS) and gallic acid (GA) are natural compounds with anti-cancer properties, and their conjugate (COS-GA) has several biological activities. Herein, the anti-cancer activity of COS-GA in SW620 colon cancer cells was investigated. MTT assay was used to evaluate cell viability after treatment with 62.5, 122, and 250 µg/mL of COS, GA, and COS-GA for 24 and 48 h. The number of apoptotic cells was determined using flow cytometry. Proteomic analysis was used to explore the mechanisms of action of different compounds. COS-GA and GA showed a stronger anti-cancer effect than COS by reducing SW620 cell proliferation at 125 and 250 µg/mL within 24 h. Flow cytometry revealed 20% apoptosis after COS-GA treatment for 24 h. Thus, GA majorly contributed to the enhanced anti-cancer activity of COS via conjugation. Proteomic analysis revealed alterations in protein translation and DNA duplication in the COS group and the structural constituents of the cytoskeleton, intermediate filament organization, the mitochondrial nucleoid, and glycolytic processes in the COS-GA group. Anti-cancer-activity-related proteins were altered, including CLTA, HSPA9, HIST2H2BF, KRT18, HINT1, DSP, and VIM. Overall, the COS-GA conjugate can serve as a potential anti-cancer agent for the safe and effective treatment of colon cancer.

Enrichment of T-cell proliferation and memory gene signatures of CD79A/CD40 costimulatory domain potentiates CD19CAR-T cell functions.

CD19 chimeric antigen receptor (CAR) T-cells have demonstrated remarkable outcomes in B-cell malignancies. Recently, the novel CD19CAR-T cells incorporated with B-cell costimulatory molecules of CD79A/CD40 demonstrated superior antitumor activity in the B-cell lymphoma model compared with CD28 or 4-1BB. Here, we investigated the intrinsic transcriptional gene underlying the functional advantage of CD19.79A.40z CAR-T cells following CD19 antigen exposure using transcriptome analysis compared to CD28 or 4-1BB. Notably, CD19.79A.40z CAR-T cells up-regulated genes involved in T-cell activation, T-cell proliferation, and NF-κB signaling, whereas down-regulated genes associated with T-cell exhaustion and apoptosis. Interestingly, CD19.79A.40z CAR- and CD19.BBz CAR-T cells were enriched in almost similar pathways. Furthermore, gene set enrichment analysis demonstrated the enrichment of genes, which were previously identified to correlate with T-cell proliferation, interferon signaling pathway, and naïve and memory T-cell signatures, and down-regulated T-cell exhaustion genes in CD79A/CD40, compared with the T-cell costimulatory domain. The CD19.79A.40z CAR-T cells also up-regulated genes related to glycolysis and fatty acid metabolism, which are necessary to drive T-cell proliferation and differentiation compared with conventional CD19CAR-T cells. Our study provides a comprehensive insight into the understanding of gene signatures that potentiates the superior antitumor functions by CD19CAR-T cells incorporated with the CD79A/CD40 costimulatory domain.

Serum Proteomic Profiling Reveals Differentially Expressed IGHG3 and A1AG1 as Potential Predictors of Chemotherapeutic Response in Advanced Non-small Cell Lung Cancer.

BACKGROUND: This study aimed to identify differentially expressed proteins in the serum of advanced non-small cell lung cancer (NSCLC) patients responding to carboplatin (CAR) plus paclitaxel (PTX) chemotherapy compared to non-responders. MATERIALS AND METHODS: Serum from 8 responders and 6 non-responders was subjected to proteomic analysis by label-free liquid chromatography tandem mass spectrometry and validated by western blotting. CAR/PTX-resistant human H1792 and A549 cells were used for evaluating gene expression. RESULTS: Fifty-two proteins were differentially expressed between responders and non-responders. Alpha 1 antitrypsin antibody, alpha 1 acid glycoprotein (A1AG1), afamin, protein S100-A9 and immunoglobulin heavy constant gamma 3 (IGHG3) were validated. IGHG3 was elevated (p=0.037) while A1AG1 was reduced (p=0.003) in responders as compared to non-responders. Gene expression of IGHG3 and ORM1 in resistant cells showed consistent results with the proteomics profiles. CONCLUSION: Serum expression levels of IGHG3 and A1AG1 proteins may be useful to recruit an NSCLC subpopulation that can benefit from CAR plus PTX standard therapy.