Project description:Although CD19 CAR T therapy has attained encouraging clinical outcomes worldwide, leukemia relapse after this therapy is associated with particular poor prognosis and has become an urgent problem to be solved. In consideration of the possible genetic or transcriptomic mechanisms underlying relapse, leukemia samples before CAR T cell infusion and after relapse were subjected to transcriptome sequencing
Project description:Cytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS. We examined CD22 CAR T-cells using the Nanostring nCounter platform and found that the expression of the PFKFB4 gene and glycolytic pathway activity were upregulated in CAR T-cells in those who developed CRS compared to those who did not have CRS.
Project description:Chimeric antigen receptor (CAR) T cells are effective against B-cell malignancies but are associated with cytokine-driven inflammatory toxicities such as cytokine release syndrome (CRS). Historically, T cell receptor (TCR) engineered T cell therapies are rarely associated with CRS. However, it is challenging to compare diverse cell products utilized in different clinical contexts and against different antigens. In this study, TCR and CAR were made to target the same source of antigen, CD22, expressed by B-cell malignancies: the TCR recognizes CD22-derived peptide processed and presented in the context of HLA-A*02:01, and the CAR recognizes CD22 protein expressed on the cell surface. In vivo studies comparing the CD22 TCR-T cells to CD22 CAR-T cells demonstrated that the TCR-T cells could clear leukemia without inducing systemic proinflammatory cytokine elevation, whereas CD22 CAR-T cells induced high levels of circulating proinflammatory cytokine reminiscent of CRS. T cells activated through either the TCR or CAR by an identical leukemia cell line demonstrated differential transcriptional responses. T cells activated via the CAR had disproportionate and significant upregulation of inflammatory gene sets compared to T-cells activated via the TCR.
Project description:While therapies targeting CD19 by antibodies, CAR-T cells and T cell engagers have improved the response rates in B-cell malignancies; the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19-immunotoxin. Single-cell (sc) RNAseq showed increase in transcriptionally distinct CD19low populations in resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19low resistant cells. ATAC-seq showed decreased chromatin accessibility at promoters of both CD19 and CD22 in the resistant cell populations. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with B-ALL that relapsed after CD19 CAR-T targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19low resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both BTK and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.
Project description:While therapies targeting CD19 by antibodies, CAR-T cells and T cell engagers have improved the response rates in B-cell malignancies; the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19-immunotoxin. Single-cell (sc) RNAseq showed increase in transcriptionally distinct CD19low populations in resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19low resistant cells. ATAC-seq showed decreased chromatin accessibility at promoters of both CD19 and CD22 in the resistant cell populations. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with B-ALL that relapsed after CD19 CAR-T targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19low resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both BTK and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.
Project description:Chimeric antigen receptor (CAR) T cell therapy have demonstrated remarkable success in treating B-cell malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced against antigen-low tumors. This was exemplified in a clinical trial of CD22-directed CAR T cells where post-CAR relapses were driven by a decrease in the surface expression of the CD22 antigen. To address the poor response of CAR T cells to antigen-low tumor cells, we performed global phosphoproteomics using a SILAC/mass spectrometry approach to examine signal transduction events within CAR T cells in response to high- and low-levels of CD22 antigen. Stimulation with low levels of CD22 antigen resulted in decreased activation of several canonical T cell signaling pathways in CAR T cells, suggesting poor utilization of LAT. To overcome this inefficiency of LAT activation, we designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART). ALA-CART cells demonstrated enhanced phosphorylation of LAT and restored downstream signaling in response to low levels of CD22. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the clinically-active, standard CD22-BBz CAR T cells. Compared to the standard CD22-BBz CAR T cells, ALA-CART cells exhibited transcriptional differences in the resting state reflecting a less differentiated state, which corresponded to an enrichment of T stem cell memory cells and enhanced in vivo persistence. Thus, through the identification of CAR signaling deficits, we rationally designed the ALA-CART platform which improves the sensitivity and persistence of CAR T cells to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies.
Project description:<p>Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory (r/r) large B-cell lymphoma (LBCL) results in durable response in only a subset of patients. MYC overexpression in LBCL tumors is associated with poor response to treatment. We tested whether a MYC-driven polyamine signature, as a liquid biopsy, is predictive of response to anti-CD19 CAR-T therapy in patients with r/r LBCL. Elevated plasma acetylated polyamines were associated with non-durable response. Concordantly, increased expression of spermidine synthase, a key enzyme which regulates levels of acetylated spermidine, was prognostic for survival in r/r LBCL. A broad metabolite screen identified additional markers which resulted in a 6-marker panel (6MetP) consisting of acetylspermidine, diacetylspermidine and lysophospholipids which was validated in an independent set from another institution as predictive of non-durable response to CAR T therapy. A polyamine centric metabolomics liquid biopsy panel has predictive value for response to CAR-T therapy in r/r LBCL. </p>
Project description:Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the clinical treatment of hematological malignancies due to the prominent anti-tumor effects. B-cell maturation antigen (BCMA) CAR-T cells have demonstrated promising effects in patients with relapsed/refractory multiple myeloma. However, the dynamics of CAR-T cell proliferation and cytotoxicity in a patient remains largely unexplored. Single-cell RNA sequencing samples were collected at three phases: CAR-T products before infusion, CAR-T on day 8 after infusion, and CAR-T on day 15 after infusion. After obtaining the PBMCs for each phase, CAR-T and endogenous T cells were collected by fluorescence-activated cell sorting with anti-Mouse IgG Biotin, FITC Streptavidin, and anti-human CD3 APC.
Project description:A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive tumor microenvironment (TME), which is densely populated and supported by protumoral glioma-associated microglia and macrophages (GAMs). Targeting CD47, a don't-eat-me signal overexpressed by tumor cells, disrupts the CD47-SIRPalpha axis and induces GAM phagocytic function. However, antibody-mediated CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. To overcome these limitations, we combined local CAR T cell therapy with paracrine GAM modulation to effectively eliminate GBM. To this end, we engineered a new CAR T cell against epidermal growth factor receptor variant III (EGFRvIII) that constitutively secretes a signal regulatory protein gamma (SIRPgamma)-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII+ GBM in a dose-dependent manner in vitro and eradicated orthotopically xenografted EGFRvIII-mosaic GBM by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon tumor re-challenge. Combining anti-CD47 antibodies with anti-EGFRvIII CAR T cells failed to achieve a similar therapeutic effect, underscoring the importance of sustained paracrine GAM modulation. Multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry on GBM-xenografted brains showed that anti-EGFRvIII-SGRP CAR T cells accelerated GBM clearance, increased CD68+ cell trafficking to tumor scar sites and promoted GAM-mediated tumor cell uptake. In a peripheral lymphoma mouse xenograft model, anti-CD19-SGRP CAR T cells had superior efficacy to conventional anti-CD19 CAR T cells. Validation on human GBM explants revealed that anti-EGFRvIII-SGRP CAR T cells had a similar tumor-killing capacity to anti-EGFRvIII CAR monotherapy but showed a slight improvement in the maintenance of tumor-infiltrated CD14+ cells. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate immune TME. This results in the additive elimination of bystander EGFRvIII- tumor cells in a manner that overcomes the main mechanisms of CAR T cell therapy resistance, including tumor innate immune suppression and antigen escape.