Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications

Abstract

Gene therapy to achieve in vivo secretion of recombinant anti-CD3 x anti-tumor bispecific antibodies in cancer patients is being explored as a strategy to counterbalance rapid renal elimination, thereby sustaining levels of bispecific antibodies in the therapeutic range. Here, we performed a comparative analysis between single- and two-chain configurations for anti-CD3 x anti-CEA (carcinoembryonic antigen) bispecific antibodies secreted by genetically-modified human cells. We demonstrate that tandem single-chain variable fragment (scFv) antibodies and two-chain diabodies are expressed as soluble secreted proteins with similar yields. However, we found significant differences in their biological functionality (i.e., antigen binding) and in their ability to induce non-specific T cell activation. Whereas single-chain tandem scFvs induced human T cell activation and proliferation in an antigen-independent manner, secreted two-chain diabodies exerted almost no proliferative stimulus when human T cells were cultured alone or in co-cultures with CEA negative cells. Thus, our data suggest that two-chain diabodies are preferable to single-chain tandem scFvs for immunotherapeutic strategies comprising in vivo secretion of bispecific antibodies aiming to recruit and activate anticancer specific lymphocytic effector T cells.

Heat Shock Protein 60 in Eggs Specifically Induces Tregs and Reduces Liver Immunopathology in Mice with Schistosomiasis Japonica

“…To investigate Treg proliferation induced by SjHSP60 in vitro , total spleen cells or CD4 + CD25 + Tregs purified from mice deficient for TLR2, TLR4 or control littermates, were cultured alone or with 0.5 μg/ml SjHSP60 or 1 μg/ml anti-CD3 mAb (BD Bioscience) as a proliferation control [ 30 , 31 ]. After 3 days, spleen cells were collected and analyzed for Ki-67 expression in CD4 + CD25 + Tregs by FCM.…”

Trial watch: Naked and vectored DNA-based anticancer vaccines

“…Along Bilusic and colleagues (National Cancer Institute, National Institutes of Health, Bethesda, MD, USA) ran a Phase I clinical trial to test the safety and efficacy of a heat-killed Saccharomyces cerevisiae strain genetically modified to express carcinoembryonic antigen (CEA), a TAA that is overexpressed by several epithelial cancers, [95][96][97] in adult individuals with metastatic, CEA-expressing carcinomas (NCT00924092). 33 Twenty-five patients were enrolled in the study and allocated to receive the vaccine (named GI-6207) s.c. every 2 weeks (we) for 3 months (mo) and then monthly thereafter.…”

In vivo secretion of anti-CD3 × anti-tumor bispecific antibodies by gene-modified cells: over a decade of T-cell engagement

“…Since then, we have published 12 scientific (original and review) articles [3][4][5][6][7][8][9][10][11][12][13][14] in which we have validated this bispecific antibodybased gene therapy strategy using different formats of bispecific antibodies (diabody and BiTE), various types of cell carriers (human T-cell lines and primary T cells, human mesenchymal and hematopoietic stem cells, and human endothelial cells), several gene transfer systems (plasmids and lentiviral vectors), and several mouse cancer models. [3][4][5][6][7][8][9][10][11][12][13][14] In 2007 we reported in Cancer Gene Therapy the usefulness of lentiviral vectors for the sustained expression of a bispecific anti-CD3 = anti-CEA diabody in human primary peripheral blood T lymphocytes. 7 In this paper we specifically stated that the recruitment of both gene-modified and nonmodified T cells, present at the tumor site, would amplify the effector response.…”

“…Given the wide availability of free Internet-based search engines, we regret the omission of the above-mentioned pioneering works, all of them indexed in PubMed, [2][3][4][5][6][7][8][9][10][11][12][13][14] showing the potential of in vivo secreted anti-CD3 = anti-(tumorassociated antigen) bispecific antibodies for cancer immunotherapy.…”