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.

Keywords: bispecific antibody; diabody; gene therapy; in vivo secretion; tandem-scFv.

Figure 1. Schematic representation of the anti-CD3 x anti-CEA bispecific antibody gene constructs. (A–B) Recombinant bispecific antibodies, including the two-chain diabody (A) and tandem single-chain variable fragment (B) gene constructs were placed under the control of the cytomegalovirus (CMV) promoter/enhancer and a heterologous signal peptide from the Oncostatin M (white box) gene. The silver boxes represent the linker peptide (G₄S) and the dark-green and yellow boxes represent 6xHis tag and myc tag respectively. The internal ribosome entry site (IRES) sequence from the encephalomyocarditis virus (EMCV) provides for expression of both diabody chains from the same CMV promoter/enhancer element. Shown are the arrangements of immunoglobulin variable heavy chain (V_H) and immunoglobulin variable light chain (V_L) domains in the two-chain diabody (A) and in the single-chain tandem scFvs (ta-scFv-A and ta-scFv-B) (B).

Figure 2. Characterization of secreted bispecific antibodies. (A) Engineered, bispecific antibodies secreted into the conditioned medium of stably transfected HEK293 cells (293^diabody, 293^ta-scFv-A or 293^ta-scFv-B) were characterized for expression levels and binding properties. (A) western blot analysis. Migration distances of molecular mass markers are indicated (kDa). The blot was developed with anti-His tag mAb. (B) The functionality of secreted antibodies was demonstrated by ELISA against plastic immobilized BSA and human CEA. (C) Flow cytofluorimetric analysis of binding of secreted bispecific antibodies to the surface of MKN45 (CEA⁺) and Jurkat (CD3⁺) cells. HeLa (CEA^–) and U937 (CD3^–) cells were used as negative controls. The y-axis shows the number of cells and the x-axis represents the intensity of fluorescence, expressed on a logarithmic scale.

Figure 3. Activation of human T cells by secreted bispecific antibodies. (A) Flow cytofluorimetric analysis of CD69 expression on Jurkat cells cultured alone or co-cultured with target cells [HeLa (CEA^–) or MKN45 (CEA⁺)] at an effector (E) to target (T) cell of 4:1, in the presence of purified anti-CD3 mAb OKT3, or fresh cell-free conditioned medium containing diabody or tandem single chain variable fragment (ta-scFv-A or ta-scFv-B) from 293^diabody, 293^ta-scFv-A, 293^ta-scFv-B, respectively. (B) ³H-Thymidine proliferation assay with human peripheral blood mononucleated cells (PBMCs) cultured alone (left panel), or co-cultured (E:T= 4:1) with irradiated HeLa (CEA^–) or MKN45 (CEA⁺) cells, in the presence of fresh cell-free conditioned medium containing diabody (gray column), ta-scFv-A (black column) or ta-scFv-B (white column). Data are reported as fold-change induction relative to the values obtained from unstimulated cells. (C) Specific lysis of tumor cells targeted by recombinant bispecific antibodies. Luciferase-tagged tumor cell lines (MKN45^Luc and Hela^Luc) were incubated with PBMC at various E to T ratios (as indicated on the x-axis) in the presence of fresh cell-free conditioned medium containing diabody (gray circles), ta-scFv-A (black circles) or ta-scFv-B (white circles). After 48 h, 20 μg/well D-luciferin was added and bioluminescence detected via luminometry. Percent viability was calculated relative to the luminescence from an equal number of input control cells and used to calculate percent specific lysis. Results are expressed as a mean ± SD (n = 3) from 1 of at least 3 separate experiments. Significance was measured by Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4. Characterization of purified bispecific antibodies. (A–C) Engineered, bispecific antibodies, including two-chain diabody and tandem, single-chain variable fragment (ta-sc-Fv), secreted into the conditioned medium of stably transfected HEK293 cells (293^diabody, 293^ta-scFv-A, or 293^ta-scFv-B) were purified by affinity column chromatography and characterized for protein M.W. and antigen binding properties. (A) Reducing SDS-PAGE of purified diabody, ta-scFv-A and ta-scFv-B antibodies. (B) Antibody titration ELISA was performed against plastic immobilized human CEA using anti-c-myc mAb. (C) Specific binding of purified bispecific antibodies (at a concentration of 1 μg/mL) to CEA and CD3 antigens expressed on the cell surface (of MKN45 and Jurkat cells, respectively) was assessed by immunofluorescence staining and flow cytometry. Anti-human CD3ε and anti-human CD66e/CEA native mAbs were used as positive controls. The y-axis shows the number of cells, while the x-axis represents the intensity of fluorescence, expressed on a logarithmic scale.