Induction of tumor immunity by autologous B lymphoma cells expressing a genetically engineered idiotype

Abstract

A fusion protein containing a B cell lymphoma idiotype (Id) variable genes from the tumor and the preparation of and granulocyte-macrophage colony-stimulating factor tumor-specific vector constructs. The low production of (GM-CSF) is a potent stimulator of tumor immunity. In three Id/GM-CSF fusion proteins by transfected cells, which is different tumor models we show that immunization with a major obstacle in the use of purified fusion proteins for autologous lymphoma cells that have been engineered to immunotherapy, is due to the presence of the cytokine express the Id in the context of GM-CSF is much more gene in the immunoglobulin locus. Low production, howeffective than immunization with an equivalent dose of the ever, is not limiting in the cell-based setting, because upon purified protein. The lymphoma Id could be modified by in vivo administration of the modified autologous cells, introducing the GM-CSF gene into the immunoglobulin (Ig) even minute expression levels are sufficient to induce heavy chain locus via gene targeting. This approach cirtumor immunity. cumvents the isolation of the rearranged immunoglobulin

B-cell lymphoma idiotypes chimerized by gene targeting can induce tumor immunity

“…Transformants having undergone site-specific recombination were identified and characterized by enzyme-linked immunosorbent assay (ELISA) using a polyclonal goat anti-human IgGFc antibody (Ab) (Dianova, Hamburg, Germany), the 38C13-Id-specific mAb E4, 26 or the A20-Id-specific mAb 6C10 20 as capturing Abs. After incubation with culture supernatants or purified proteins, bound Ig was detected with peroxidase-labeled anti-human IgGFc (Dianova).…”

“…The cells were transfected with the expression vector p3159 or BCMGSNeo-V H A20-hu␥1-GMCSF, respectively, which contained the 38C13 or A20 V H gene segment and the human IgG1 C region, which was fused in frame to GM-CSF. 20 In the transfectants 1H7 (derived from 38C13) and 5C12 (A20-derived), the transferred IgH chains were correctly assembled with the endogenous Ig chains and secreted together with the parental Ig. The expression levels (250 and 25 ng/10 6 cells/24 hours for 1H7 and 5C12, respectively, as determined in culture supernatants and after purification by protein A chromatography) as well as the integrity of the fusion products were demonstrated by ELISA 20 using the anti-38C13-Id Ab E4 26 or the anti-A20-Id Ab 6C10, 20 anti-human IgGFc Ab, and anti-GM-CSF mAb.…”

“…p3159 (a gift from R. Levy, Stanford University, Stanford, Calif 17 ) and BCMGSNeo-V H A20-hu␥1-GMCSF 20 are expression vectors that contain the 38C13 and A20 V H gene, respectively, the human IgG1 C region fused to GM-CSF, and the gpt gene as a selection marker. The recombination vectors pSVgpt-hu␥1-A4, pSVgpt-hu␥1-A5, and pSVgpt-hu␥1-A6 contain a homology flank comprising 2.3, 3.0, and 5.8 kb, respectively, derived from the murine Ig intron, the human IgG1 C gene segments, and the gpt gene.…”

“…The rat hybridoma 6C10 secretes a mAb with specificity against the A20 Id. 20 All cell lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 M 2-mercaptoethanol at 37°C. For transfection, 20 g of the recombination vector was linearized with EcoRI (which cuts within the homology region) or BamHI and mixed with 10 6 cells in 700 L of RPMI 1640.…”

“…17 We have shown elsewhere that the effect of soluble modified Id proteins is significantly enhanced when autologous lymphoma cells engineered to express the modified Id are used as a vaccine instead of the protein preparation. 19,20 Here we show that a xenogeneic Fc region can indeed induce tumor immunity when presented by autologous lymphoma cells whose Id is solely chimerized by substituting the murine IgH constant (C) exons with a human isotype by gene targeting. 21,22 …”