Chimeric anti-CD19 Research Articles

Cost-Effectiveness of brexucabtagene autoleucel for relapsed/refractory mantle cell lymphoma

Brexucabtagene autoleucel is a chimeric anti CD19 antigen receptor T-cell therapy that allows durable responses in relapsed/refractory (R/R) mantle cell lymphoma (MCL). The present study compared the clinical and economic outcomes of R/R MCL patients (pre-exposed to ibrutinib and chemoimmunotherapy) treated with brexucabtagene autoleucel versus Rituximab, bendamustine, cytarabine (R-BAC) in the Italian Healthcare System. A partitioned-survival model extrapolated survival and healthcare costs of R/R MCL patients over a lifetime horizon. Discounted and quality-adjusted life expectancy (QALY) was 6.40 versus 1.20 for brexucabtagene autoleucel versus R-BAC and lifetime costs were €411,403 versus €74,415, respectively, which corresponds to a cost of €64,798 per QALY gained. The results were highly sensitive to brexucabtagene autoleucel acquisition cost and to assumptions on long-term survival, therefore the cost-effectiveness of brexucabtagene autoleucel for patients with R/R MCL requires validation with longer follow-up data and in specific risk subgroups.

Dual Targeting of Mesothelin and CD19 with Chimeric Antigen Receptor-Modified T Cells in Patients with Metastatic Pancreatic Cancer

B cells infiltrate pancreatic ductal adenocarcinoma (PDAC) and in preclinical cancer models, can suppress Tcell immunosurveillance in cancer. Here, we conducted a pilot study to assess the safety and feasibility of administering lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous Tcells redirected against mesothelin to target tumor cellsalong with CART cells redirected against CD19 to deplete B cells. Both CARs contained 4-1BB and CD3ζ signaling domains. Three patients with chemotherapy-refractory PDACreceived 1.5 g/m2 cyclophosphamide prior to separate infusions of lentiviral-transduced Tcells engineered to express chimeric anti-mesothelin immunoreceptor SS1 (CART-Meso, 3× 107/m2) and chimeric anti-CD19 immunoreceptor (CART-19, 3× 107/m2). Treatment was well tolerated without dose-limiting toxicities. Best response was stable disease (1 of 3 patients). CART-19 (compared to CART-Meso) cells showed the greatest expansion in the blood, although persistence was transient. B cells were successfully depleted in all subjects, became undetectable by 7-10days post-infusion, and remained undetectable for at least 28days. Together, concomitant delivery of CART-Meso and CART-19 cells in patients with PDAC is safe. CART-19 cells deplete normal B cells but at the dose tested in these 3 subjects did not improve CART-Meso cell persistence.

Adoptive Cell Therapy: A Novel and Potential Immunotherapy for Glioblastoma.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with very poor prognosis and few advances in its treatment. Recently, fast-growing cancer immunotherapy provides a glimmer of hope for GBM treatment. Adoptive cell therapy (ACT) aims at infusing immune cells with direct anti-tumor activity, including tumor-infiltrating lymphocyte (TIL) transfer and genetically engineered T cells transfer. For example, complete regressions in patients with melanoma and refractory lymphoma have been shown by using naturally tumor-reactive T cells and genetically engineered T cells expressing the chimeric anti-CD19 receptor, respectively. Recently, the administration of ACT showed therapeutic potentials for GBM treatment as well. In this review, we summarize the success of ACT in the treatment of cancer and provide approaches to overcome some challenges of ACT to allow its adoption for GBM treatment.

Abstract 735: SAR of amanitin and optimization of linker-amanitin derivatives for solid tumors

Abstract Background: ATACs (Antibody Targeted Amanitin Conjugates) comprise a new class of antibody-drug conjugates using amanitin as toxic payload. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription process at very low concentrations. We accomplished the chemical synthesis of amanitin and were able to synthesize amanitin variants in order to optimize the toxin structure for different tumors and antibodies. We will present in vitro and in vivo data of eight different linker-amanitin constructs attached to three different antibodies targeting solid tumors. Material and methods: Cell lines: JIMT-1, SKBR-3, BT474 and NCI-N87 (used for anti-Her-2-ATACs); LnCap, 22RV1, MDA-PCa2b and C4.2 (used for anti-PSMA-ATACs); Raji, Raji Luc, Nalm-6 and MEC-2 (used for anti-CD19-ATACs) Antibodies: Anti-Her-2 (cysteine engineered monoclonal antibody, Heidelberg Pharma); humanized anti-PSMA (Albert Ludwig University Freiburg, medical center; humanization at Lonza Group AG; cysteine engineered monoclonal antibody, Heidelberg Pharma); chimeric anti-CD19 (DKFZ Heidelberg, Germany; cysteine engineered monoclonal antibody, Heidelberg Pharma). Toxic warhead: Cysteine reactive linker-amanitin constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibodies. Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega). Animal models: Subcutaneous Mouse xenograft tumor models (Her-2-, PSMA- and CD19-positive cell lines) were performed in single-dose experiments. Tolerability was assessed in mice and will be assessed in non-human primates (NHP). Results: Eight different amanitin-linker constructs were synthesized. They differed in the attachment site of the linker at the amanitin as well as in the toxin core structure. All ATACs showed in vitro cytotoxicity on target positive cell lines in the picomolar range. In mouse xenograft models, ATACs with four of the eight linker-amanitin derivatives caused dose-dependent tumor regression and complete remission after a single i.v. dose of 2.0 mg/kg in s.c. xenografts irrespective of the antibody and target used. In contrast the other four linker-amanitin derivatives were only poorly effective in vivo while showing comparable in vitro activities. When comparing subcutaneous and intravenous xenograft models using the same cancer cell line, the different efficacy of the eight linker-amanitin variants was only detected in subcutaneous but not in intravenous xenografts. Mouse tolerability studies of ATACs showed a MTD of at least 10mg/kg for all linker-amanitin variants. Conclusions: Different efficacy of linker-amanitin derivatives with regard to mouse xenograft models was detected. An SAR profile of amanitin could be established which enabled the selection of optimized linker-amanitin variants for the use of ATACs in solid tumors. Citation Format: Michael Kulke, Anikó Pálfi, Christoph Müller, Werner Simon, Susanne Werner-Simon, Christian Lutz, Torsten Hechler, Andreas Pahl. SAR of amanitin and optimization of linker-amanitin derivatives for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 735.

Pilot Study of Non-Viral, RNA-Redirected Autologous Anti-CD19 Chimeric Antigen Receptor Modified T-Cells in Patients with Refractory/Relapsed Hodgkin Lymphoma (HL)

Pilot Study of Non-Viral, RNA-Redirected Autologous Anti-CD19 Chimeric Antigen Receptor Modified T-Cells in Patients with Refractory/Relapsed Hodgkin Lymphoma (HL)

Upregulation of Adhesion Molecules on Leukemia Targets Improves the Efficacy of Cytotoxic T Cells Transduced With Chimeric Anti-CD19 Receptor

T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.

Abstract 3511: Genetically engineered natural killer (NK) cell immunotherapy for poor risk B-cell (CD20+) leukemia and lymphoma (L/L)

Abstract Background: Patients with CD20+ L/L at relapse have a chemotherapy resistant phenotype and a dismal prognosis (Cairo et al, Blood, 2007). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Beziat V et al, Leukemia, 2009) but cell number and tumor recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Ex vivo expansion of PBNK cells with genetically engineered K562-mbIL15-41BBL cells (modified K562) and the expanded PBNK cells transduced with chimeric anti-CD19 antigen receptor (CAR) have been previously reported (Imai C et al, Blood. 2005). Objective: We investigated the functional activities and cytolytic effect of chimeric anti-CD20 antigen receptor (CAR+) engineered PBNK cells expanded with K562-mbIL15-41BBL cells (modified K562) against CD20+ L/L both in vitro and in vivo. Methods: Whole peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. PBMC were incubated with mitomycin C modified K562 cells in culture medium with 10 IU/ml IL-2 for 7 or 14 days. CD56 and CD3 expression were evaluated by flow cytometry. The CAR+ was constructed in a MSCV-anti-CD20BB-CD3-zeta-GFP plasmid (generously supplied by Dario Campana, MD, PhD, St. Jude Children's Hospital). CAR+ and CAR− retroviruses were generated in 293T cells with helper plasmids. Jurkat cells were used to examine infection efficiency. Anti-CD20 CAR expression was examined by flow cytometry and by western blot of whole cell lysates for CD3zeta. PBMC stimulated with modified K562 were transduced with retroviruses as previous described (Imai C et al, Blood. 2005). PBNK cells cytotoxicity was assessed by europium release assay at 2:1 E:T ratio against CD20+ Ramos. Results: CD56+CD3− PBNK cells were expanded significantly more following modified K562 stimulation compared to media alone at day7 (60.94+ 3.63% vs 8.05+0.49%, n=6, P<0.001). CD56−CD3+ PBT cells were significantly reduced compared to media alone at day7 (22.08+2.22% vs 75.73+0.75%, n=6, P<0.001). CAR+ and CAR− retrovirus supernants infected Jurkat cells at 40%-97% efficiency and infected expanded PBMC at 1%-10% range (n=3). The anti-CD20 CAR expression was further confirmed by flow cytometry and western blot. We also observe that cytotoxicity was enhanced with anti-CD20 CAR+ PBNK compared to CAR− PBNK (41+ 1.1% vs 24.5+ 3.7%, n=3) against Ramos at effector to target ratio 2:1. Conclusion and Future Directions: PBNK can be significantly induced following modified K562 stimulation. Anti-CD20 CAR enhances PBNK anti-tumor activity against CD20+ Ramos. Future directions include further characterizing the cytotoxicity activity of Anti-CD20 CAR engineered expanded PBNK cells against L/L in-vitro and survival in xenogafted mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3511. doi:1538-7445.AM2012-3511

Cytotoxic T cells transduced with chimeric anti-CD19 receptors prevent engraftment of primary lymphoblastic leukemia in vivo

Cytotoxic T cells transduced with chimeric anti-CD19 receptors prevent engraftment of primary lymphoblastic leukemia in vivo

Expression of a functional mouse-human chimeric anti-CD19 antibody in the milk of transgenic mice

Human B cell lymphomas are suitable targets for immunotherapy. Clinical trials with mouse-human chimeric B cell-specific monoclonal antibodies (mAbs) have already shown promising results. However, limitations for their use in clinical trials can be the lack of sufficient amounts and high production costs. Expression of mAbs in the mammary gland of transgenic animals provides an economically advantageous possibility for production of sufficient quantities of a promising antibody for clinical trials and beyond. In this paper, we show the feasibility of this approach, by generating transgenic mice expressing mouse-human chimeric anti-CD19 mAbs in their milk. Mouse anti-CD19 variable (V) region genes were combined with human IgG1 heavy (H) and kappa light (L) chain constant (C) region genes and fused to the bovine β-lactoglobulin (BLG) promoter in two separate expression cassettes. Co-injection resulted in five transgenic lines. In one of these lines completely assembled chimeric mAbs were secreted into the milk, at an approximate level of 0.5mg/ml. These mAbs were able to bind specifically to the CD19 surface antigen on human B cells.

In vitro and in vivo antitumour activity of a chimeric anti-CD19 antibody

In vitro and in vivo antitumour activity of a chimeric anti-CD19 antibody

In vitro and in vivo antitumor activity of a chimeric anti-CD19 antibody.

Mouse monoclonal antibodies to CD19 detect an antigenic determinant expressed exclusively on the surface of B lymphocytes, and have previously been shown to be potentially useful therapeutic reagents for human B cell lymphoma. We report the production and characterization of a mouse/human chimeric antibody, cCD19, with potent in vivo antitumour activity. The genes encoding the variable domains for heavy (VH) and light (VL) chains were subcloned into eukaryotic expression vectors containing human constant region genes (IgG1 and kappa), and co-transfected into non-secreting Sp2/0 mouse myeloma cells. Intraperitoneal administration of cCD19 produced inhibition of growth of subcutaneous CD19+ Sultan human B lymphoma tumours in scid/scid mice. When the antibody was administered 18 and 20 days after subcutaneous tumour inoculation, an approximately 30% reduction in tumour size was noted by day 29. cCD19 faithfully mimicked the in vitro binding characteristics of mCD19 as (a) the chimeric antibody was shown by flow cytometry to bind exclusively to cell lines that expressed CD19, (b) cCD19 was able to inhibit the binding of mCD19 on CD19+ cells completely and (c) the affinity of binding of the two antibodies was not significantly different [Ka = (2.03 +/- 1.5) x 10(8)]. In bio-distribution studies, up to 14.8% of the total injected antibody dose per gram of tissue was localized in CD19+ Sultan tumours at 24 h approximately, 14.4% was present in the tumors at 48 h, and about 13.7% at 72 h. These levels were comparable to mCD19 administered in the same fashion. cCD19 conjugated to idarubicin was specifically and strongly cytotoxic to CD19+ cells cultured in vitro, and demonstrated an IC50 of 0.17 microM, similar to that of mCD19 (0.32 microM) and approximately 14-fold greater than the IC50 of free idarubicin. The specific cytotoxic capacity of cCD19 and its likely reduced immunogenicity suggest that it may potentially be of use in the treatment of refractory B cell lymphoma in humans.