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

Abstract

Infiltrating T lymphocytes are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of defective T-cell activation events. Recently, we described a strategy for the tumor-specific polyclonal activation of tumor-resident T lymphocytes based on the in situ production of recombinant bispecific antibodies (bsAbs) by transfected nonhematological cell lines. Here, we have constructed a novel HIV-1-based lentiviral vector for efficient gene transduction into various human hematopoietic cell types. Several myelomonocytic and lymphocytic cell lines secreted the anticarcinoembryonic antigen (CEA) Â anti-CD3 diabody in a functionally active form with CD3 þ T-cell lines being the most efficient secretors. Furthermore, primary human peripheral blood lymphocytes (PBLs) were also efficiently transduced and secreted high levels of functional diabody. Importantly gene-modified PBLs significantly reduced in vivo tumor growth rates in xenograft studies. These results demonstrate, for the first time, the utility of lentiviral vectors for sustained expression of recombinant bsAbs in human T lymphocytes. Such T lymphocytes, transduced ex vivo to secrete the activating diabody in autocrine fashion, may provide a promising route for a gene therapy strategy for solid human tumors.

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“…In fact, we have previously shown that human T lymphocytes, transduced ex vivo to secrete the aCEA/aCD3 diabody in autocrine manner, inhibited the growth of CEA-positive tumors. 8 However, the short life span of activated human T lymphocytes and their inefficient transduction constitute important drawbacks for their application in cancer therapy.…”

“…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.…”