CD20 is a molecular target for scFvFc:zeta receptor redirected T cells: Implications for cellular immunotherapy of CD20+ malignancy

Abstract

The CD20 molecule was evaluated as a B-cell lymphoma target epitope for T cells expressing a CD20-specific single-chain FvFc-zeta (scFvFc:zeta) chimeric receptor. A cDNA construct consisting of a murine kappa leader sequence, CD20-specific scFv, human immunoglobulin (Ig) G1 hinge-C(H)2-C(H)3, the human CD4 transmembrane, and the intracellular signaling domain of the human CD3 complex's zeta chain was synthesized by polymerase chain reaction splice-overlap extension. The human CD4+ Jurkat cell line was electroporated with the CD20-specific scFvFc:zeta construct cloned into the mammalian expression vector pcDNAneo. Western blot analysis of transfectant whole cell lysate with an anti-zeta antibody demonstrated the expression of both endogenous zeta and the chimeric receptor protein, with a mobility consistent with the expected molecular weight of 66 kD under reducing conditions; nonreduced lysate revealed a chimeric receptor complex of approximately 132 kD. The scFvFc:zeta receptor was present on the cell surface as detected by flow cytometry of T-cell transfectants stained with an anti-mouse Fab-specific antibody and anti-human Fc gamma-specific monoclonal antibody. Coculture of Jurkat transfectants with CD20+ lymphoma cells resulted in the accumulation of interleukin (IL)-2 in culture supernatants as detected by ELISA. IL-2 production was triggered by the specific interaction between the CD20 molecule and the scFvFc:zeta as IL-2 was not detected in cultures with mock transfected Jurkat cells or CD20- stimulator cells. Furthermore, IL-2 production was inhibited by the addition of a soluble anti-CD20 monoclonal antibody to cocultured Jurkat transfectants and CD20+ stimulator cells. The capacity of CD20 to trigger the lytic machinery of scFvFc:zeta-expressing cytotoxic T lymphocytes (CTLs) was assessed using the murine allo-specific CD8+ CTL clone 2c. CD20-specific redirected cytolytic activity against human lymphoma targets was observed with 2c transfectants in a 4-hour chromium release assay. These results demonstrate that CD20 can serve as a target epitope for scFvFc:zeta receptor-expressing T cells.

Tumor-Derived Chemokine MCP-1/CCL2 Is Sufficient for Mediating Tumor Tropism of Adoptively Transferred T Cells

“…The protein fusion between ffLuc and the zeocin resistance gene dictates that all zeocin-resistant cells also express the ffLuc reporter gene. Firefly luciferase-positive T cells were propagated ex vivo on a 14-day stimulation cycle with 30 ng/ml OKT3, a 5:1 ratio of irradiated allogeneic PBMC and EBV-transformed lymphoblastoid (LCL) feeder cells, and 25 U/ml rhIL-2 (Chiron) as described previously (24). T cells were cultured in RPMI 1640 (Irvine Scientific) with 10% FCS (HyClone), 25 mM HEPES (Irvine Scientific), 2 mM glutamine (Cambrex), and fresh rhIL-2 was added to cultures every 48 h. All functional assays were performed with T cell effectors between days 10 and 14 from stimulation with OKT3.…”

Genetic modification of human T lymphocytes for the treatment of hematologic malignancies

“…For hematologic malignancies, CAR technology allows targeting of lineage restricted antigens, such as CD19 42 and CD20 43 that are conserved in the malignant counterpart, by clinical trials have been registered in the USA for infusing T lymphocytes expressing CD19-or CD20-specific CARs in patients with B-cell derived malignancies, 44 and results from many of these studies have recently been reported (Table 2). 5,10,[45][46][47][48][49] When analyzing the results of these trials, it is important to appreciate that few patients have so far been enrolled in each study, and each study cohort is heterogeneous with regards to patient age and type/stage of disease.…”

Immunotherapy for B-Cell Neoplasms Using T-Cells Expressing Chimeric Antigen Receptors : From Antigen Choice to Clinical Implementation

“…In early ex vivo studies, both T cells expressing CAR19 (CART19) and CART20 efficiently lysed a wide panel of tumour cell lines expressing CD19 and CD20 antigens, respectively, as well as B cells freshly isolated from patients with B-cell neoplasms. [50][51][52][53][54][55][56] In animal models, CART19 and CART20 also eradicated systemic cell tumours established in severe combined immunodeficient (SCID) mice that were expressing CD19 or CD20. 51,52 These preclinical experiments have supported the notion that under appropriate conditions, T cells can be therapeutically redirected to eliminate malignant B cells and have justified the translation of these genetically engineered T cells to clinical settings.…”