S. Grupp

Cell Division Rates of Primary Human Precursor B Cells in Culture Reflect In Vivo Rates

Bone marrow stroma‐based cultures provide a powerful model for studying cell division and apoptosis of primary human precursor B cells. Studies using this model are elucidating the mechanisms by which stromal cells inhibit apoptosis of cultured normal precursor B cells and have demonstrated that the apoptotic rate of cultured leukemic precursor B cells can predict clinical outcome in acute lymphoblastic leukemia. In contrast to apoptosis, cell division in this model has not been well characterized. In this study, we quantified the rates of cell division in cultured primary human normal and leukemic precursor B cells by labeling precursor B cells with the fluorescent dye carboxyfluorescein diacetate, succinimyl ester. Based on the rate of decreasing fluorescent signal over 3 weeks, normal CD19+, CD10+ precursor B cells divided once every 90.5 hours, a number that correlates well with the known in vivo rate of 65.5 hours. The division rates were similar among different cultures and constant throughout the 3 weeks of culture, suggesting that the variable expansions of precursor B cells seen among different samples and culture durations are not secondary to different cell division rates. Unlike normal cells, cultured leukemic B cells had a heterogeneous division rate that ranged from once every 26–240 hours. These rates correlated well with their respective in vivo proliferation index. These findings indicate that the stroma‐based cultures faithfully replicate in vivo cell division rates and can be used to elucidate the pathways that regulate cell division of primary human precursor B cells.

Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy.

The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.

Abstract 4707: Genetically engineered NY-ESO-1-specific T cells in HLA-A2+ patients with synovial sarcoma

NY-ESO-1 is expressed in ∼70% of synovial sarcomas and not expressed in vital tissues. We report interim results of NCT01343043 evaluating safety and activity of autologous T cells engineered to express an HLA-A2+ restricted, affinity-enhanced T cell receptor (TCR) targeting NY-ESO-1. HLA-A2+ patients with unresectable, metastatic, or recurrent synovial sarcoma were eligible if tumors expressed NY-ESO-1 by IHC. Lymphocytes were activated using anti-CD3/28 microbeads, genetically modified with a lentivector, then cryopreserved. Subjects received fludarabine 30mg/m2/d (D-6 to -2) and cyclophosphamide 1800mg/m2/d (D-3,-2), and infusion of engineered T cells. Systemic IL-2 was not administered. Nine subjects have received NY-ESO-1 cell infusions. Median transduced T cell dose was 3.4 × 109 cells (range 0.4-14.4), 60×106 cells/kg (range 5.7-165.5), and median transduction efficiency was 45.8%. Toxicity likely attributable to the T cells included fever, and grade 1-2 cytokine release syndrome. No autoimmune toxicity has been observed. Circulating engineered NY-ESO-1 cells were detected in all patients, peaking 3-21 days post-infusion. Persistence has been evaluated beyond 3 months in 4 subjects, all of whom had detectable NY-ESO-1 cells at 4 mos, 6 mos+, 12 mos+ and 12 mos+. We identified persisting NY-ESO-1 T cells using dextamer and/or anti-vβ13.1 mAbs, and observed that a high fraction of CD4+ and CD8+ NY-ESO-1 TCR expressing T cells were CD45RA+CCR7+CD95+, consistent with a stem cell memory phenotype. Persisting cells also demonstrate a polyfunctional (IFN-γ and TNF-α) and cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Of 8 patients whose follow-up is sufficient to assess response, 4 experienced objective responses (1CR x 9 mos, 1PR x 9 mos, 2PR x 6 mos). Tumor shrinkage could not be attributed to chemotherapy alone as progressive decreases in tumor size were observed over several months following completion of the lymphodepleting regimen. All PR patients (2 upon signs of progression and 1 still responding to therapy) ultimately underwent resection for residual disease and two remain without evidence of disease. Adoptive immunotherapy with NY-ESO-1 engineered T cells shows promising results in synovial sarcoma with acceptable toxicity. High dose IL-2 is not required for therapeutic benefit with this regimen. Citation Format: Melinda S. Merchant, Sandra P. D9Angelo, Hua Zhang, Donna Bernstein, Gwen Binder-Scholl, Tom Holdich, Luca Melchiori, Dan Williams, Marylene Fortin, Yoav Peretz, Jason Howe, Michael Mehler, Bruce A. Hug, Matthew Wright, Stephen Grupp, Paul A. Meyers, William Tap, Bent Jakobsen, Crystal L. Mackall. Genetically engineered NY-ESO-1-specific T cells in HLA-A2+ patients with synovial sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4707. doi:10.1158/1538-7445.AM2015-4707

Correction to: Grading of cytokine release syndrome associated with the CAR T cell therapy tisagenlecleucel

The original article contains several small errors. The errors & concurrent corrections are listed below [1]:

Cardiac Profile of Chimeric Antigen Receptor T Cell Therapy in Children: A Single-Institution Experience

Immunotherapy with chimeric antigen receptor (CAR)-modified T cells targeting CD19 for pediatric acute lymphoblastic leukemia (ALL) has demonstrated significant efficacy. The principle toxicity is cytokine release syndrome with resultant hypotension. However, the spectrum of cardiovascular effects associated with CAR T cell therapy has not been systematically evaluated. We reviewed all patients who received CD19-directed CAR T cells at the Childrens Hospital of Philadelphia between April 2012 and September 2016. The primary endpoint was hypotension-requiring inotropic support. Secondary endpoints included echocardiographic dysfunction at discharge and 6-month follow-up. Descriptive and univariate analyses were performed, and 98 encounters were included (55% male patients; mean age, 11.8 years [range, 1.7 to 27.1]); 98% had B-ALL. Before infusion 10 had cardiomyopathy and 1 had single-ventricle physiology. Primary endpoint occurred in 24 patients with mean onset 4.6 days (range, 1 to 9) after CAR T cell infusion, including 6 patients receiving milrinone. Worsened systolic function occurred in 10 patients; there were no cardiac-related deaths. Pretreatment factors associated with primary endpoint included higher pretreatment blast percentage on bone marrow biopsy (blast > 25%: odds ratio, 15.5; 95% confidence interval, 5.1 to 47.1; P < .001) and baseline lower ejection fraction (P = .019) or diastolic dysfunction (P = .021); neither pre-existing cardiomyopathy (P = .062), total body irradiation (P = .629), nor anthracycline dose (P = .444) were associated. At discharge, 7 patients had worsened echocardiographic function, but persistent dysfunction by the 6-month follow-up was rare. Pretreatment factors were not associated with persistent dysfunction at discharge. This is the first study to describe the cardiovascular effects of pediatric CAR T cell therapy. Although 10% had new systolic dysfunction after treatment, persistence was rare. Pretreatment blast count > 25% or pre-existing cardiac dysfunction increased the risk for hypotension-requiring inotropic support; these patients may warrant close observation.

Abstract 3143: Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope and contributes to treatment failure

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA CD19 is expressed on most B-cell neoplasms, including acute lymphoblastic leukemias (B-ALL). Chimeric antigen receptor (CAR)-armed T cells targeting CD19 (CART-19) yield complete remission rate of 70-90% in B-ALL patients; the bispecific anti-CD19/CD3 antibody blinatumomab also shows clinical efficacy. Yet relapses due to the apparent CD19 loss have been observed following both therapies. Here we investigated the molecular mechanisms of epitope loss using five B-ALL samples obtained after CD19-directed therapy. These samples were matched, when available, with CD19-positive pre-treatment leukemias. Although the CD19 epitope recognized by CART-19 was undetectable in relapsed specimens, in all cases the CD19 genomic sequence was retained and the mRNA levels were only slightly reduced, suggesting regulation at the level of transcript processing. Indeed, using RT-PCR and RNA-Seq we detected several alternatively spliced CD19 mRNA species lacking exons 5-6 and/or exon 2 (Δex2), which encodes the cognate CART-19 epitope. In the sets of matched samples, the relative abundance of the Δex2/5-6 mRNA isoforms was increased post-treatment, and accordingly truncated CD19 protein variants were detectable using antibodies recognizing alternative epitopes. In one relapsed leukemia with a nonsense mutation in exon 2 of CD19, Δex2 was the only expressed isoform. To determine the contribution of alternative splicing to immune escape, we introduced CD19-expressing retroviruses into murine neoplastic B-cells, which had lost endogenous CD19 expression following silencing of its main transcriptional regulator Pax5. While skipping of exon 2 made the resultant protein invisible to CART-19, it partly rescued the defects in cell proliferation associated with CD19 loss. Collectively, these data suggest the existence of a novel mechanism of resistance to immunotherapy, based not on mutations in the coding sequence but rather on selection for alternatively spliced target protein isoforms (e.g., CD19 Δex2). Our findings could lead to the development of additional CD19-targeting CARTs for treating relapsed patients. Citation Format: Elena Sotillo, David Barrett, Aseb Bagashev, Kathryn Black, Caludia Lanauze, Derek Oldridge, Robyn Sussman, Colleen Harrington, Elaine Y. Chung, Ted J. Hofmann, Shannon L. Maude, Nicole M. Martinez, Pichai Raman, Marco Ruella, David Allman, Elad Jacoby, Terry Fry, Yoseph Barash, Kristen W. Lynch, Crystal Mackall, John Maris, Stephen A. Grupp, Andrei Thomas-Tikhonenko. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope and contributes to treatment failure. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2015-3143