Induction of Human T Lymphocyte Cytotoxicity and Inhibition of Tumor Growth by Tumor-Specific Diabody-Based Molecules Secreted from Gene-Modified Bystander Cells

Abstract

Infiltrating T cells are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of the absence of activation and/or costimulatory signals. We describe a strategy for cellular antitumor immunotherapy by the in situ production of soluble bifunctional Ab-based molecules that activate and retarget T cells to the tumor. We genetically modified cells to simultaneously secrete two bifunctional molecules, a bispecific diabody directed against the carcinoembryonic Ag (CEA) and the CD3ε chain of the TCR (αCEA × αCD3), and a fusion protein comprising the extracellular portion of B7-1 fused to a bivalent anti-CEA diabody (B7-αCEA). Together, αCEA × αCD3 and B7-αCEA proved potent at inducing the activation, proliferation, and survival of primary human T cells. When producer cells were cocultured with primary T cells and CEA+ cancer cells, αCEA × αCD3 and B7-αCEA acted in combination to activate and retarget T cell cytotoxicity and completely abrogate tumor growth in the coculture. Furthermore, the introduction of just a few such producer cells at the tumor site efficiently inhibited the growth of established human colon carcinoma xenografts. Despite a cumbersome generation process, the use of autologous gene-modified producer cells opens the way for a new diabody-based gene therapy strategy of cancer.

Factory neovessels: engineered human blood vessels secreting therapeutic proteins as a new drug delivery system

“…11 Early passage HUVECs were transduced at a vector multiplicity of infection of 10, with lentivirus encoding the aCEA/aCD3 diabody and enhanced green fluorescent protein (EGFP) (Lenti dAb ), or with a lentivirus harboring a luciferase-IRES-EGFP cassette (Lenti Luc ). 7,8 More than 90% of transduced HUVEC (HUVEC dAb and HUVEC Luc ) expressed EGFP for at least 30 days in vitro ( Figure 1a) and showed normal morphology, phenotype and function (data not shown). Interestingly, the secretion of functional aCEA/aCD3 diabody molecules remained stable during this period with levels around 100 ng ml À1 per 10 5 cells per 72 h at day 30 (Figure 1b).…”

“…2 We have previously shown the feasibility of in vivo production and systemic delivery of therapeutic antibodies by gene-modified human cells. [6][7][8] Bispecific antibodies are non-natural immunoglobulin-based molecules that contain two distinct binding specificities. 9 Bispecific antibodies redirect the cytolytic activity of a variety of immune effector cells toward tumor cells by binding to cell activation molecules with one domain and to specific tumor-associated antigen on cancer cells with the other.…”

“…9 Bispecific antibodies redirect the cytolytic activity of a variety of immune effector cells toward tumor cells by binding to cell activation molecules with one domain and to specific tumor-associated antigen on cancer cells with the other. T-cell-activating bispecific antibodies have shown great potential for the treatment of malignant diseases in both animal models 7,8,11 and humans. 12 Recently, it was reported that systemically administered recombinant anti-CD19 Â anti-CD3 antibody induced partial and complete tumor regressions in patients with non-Hodgkin's lymphoma.…”

“…12 Recently, it was reported that systemically administered recombinant anti-CD19 Â anti-CD3 antibody induced partial and complete tumor regressions in patients with non-Hodgkin's lymphoma. 12 In previous works we have shown the antitumoral effect of a small recombinant bispecific antibody (diabody, dAb) 13,14 directed against the human carcinoembryonic antigen (CEA, CD66) and the T-cell co-receptor CD3 (anti-CEA Â anti-CD3), secreted by different human cells, including T lymphocytes 7,8 and MSCs. 10 However, activated T lymphocytes possess a short life span, and this implies an obvious limitation to their application in a gene therapy strategy.…”

Inhibition of tumor growth in vivo by in situ secretion of bispecific anti-CEA × anti-CD3 diabodies from lentivirally transduced human lymphocytes

“…5 In fact, we have demonstrated proof-of-principle for a novel genetic strategy of T-cell activation by paracrine secretion of recombinant bsAbs. 7 Bispecific aCEA (carcinoembryonic antigen) Â aCD3 diabodies were secreted at high levels by transfected nonhemaopoietic cell lines and were able to efficiently activate peripheral blood lymphocytes (PBLs) to proliferate and eliminate CEA-expressing tumor cells.…”