Generation, immunologic characterization and antitumor effects of human monoclonal antibodies for carcinoembryonic antigen.

We generated fully human mAbs (HmAbs) to carcinoembryonic antigen (CEA) using the KM mouse, which carries a human chromosome 14 fragment containing the entire Ig H chain loci and human kappa L chain segments in the mouse genome. Forty-six hybridoma clones producing HmAbs to CEA were thus obtained by fusing the P3-U1 mouse myeloma cells with splenocytes of the KM mice immunized with CEA. Among them, 22 clones produced HmAbs that reacted with CEA but not with 3 other CEA-related cell adhesion molecule (CEACAM) family members, CEACAM1, CEACAM6 and CEACAM8. In 12 HmAbs examined, 8 were IgG4, 2 were IgG3, 1 was IgG2, and the other was IgG1. The affinity constants for CEA of these HmAbs were comparable to those of the previously prepared mouse anti-CEA mAbs (MmAbs). BIAcore analyses revealed that 1 and 2 of the 22 HmAbs react with 2 epitopes defined by MmAbs on the domain N and the domain A1 or B1 of CEA, respectively. In the presence of human complement in vitro, 2 HmAbs tested showed substantial cytotoxicity, namely, 50-65%, against CEA-expressing tumor cells. With human lymphokine-activated killer cells in vitro, 3 HmAbs tested exhibited 40-65% Ab-dependent cell-mediated cytotoxicity against the tumor cells. Moreover, one of the HmAbs induced a significant inhibition of tumor growth when administered to mice xenografted with the CEA-expressing cells. Considering their lack of immunogenicity to humans, these CEA-specific HmAbs may be useful for immunotherapeutic approaches as well as for immunodiagnosis.

Targeting of interleukin-2 to human MK-1-expressing carcinoma by fusion with a single-chain Fv of anti-MK-1 antibody.

BACKGROUND: Targeting of cytokines into the tumor sites using antibody-cytokine fission proteins represents a novel approach in cancer immunotherapy. We previously reported a novel monoclonal antibody, FU-MK-1, which recognizes a glycoprotein antigen (termed MK-1 antigen) that is overexpressed on the surface of a majority of carcinomas. MATERIALS AND METHODS: To target IL-2 and cytotoxicity of effector cells to MK-1-expressing tumor cells, we genetically fused recombinant human interleukin-2 (rhIL-2) to a single chain variable fragment (scFv) antibody derived from FU-MK-1. The resulting fission protein, designated FUscFv/IL-2 was expressed in Pichia pastoris, purified by Ni-affinity chromatography, and characterized for the MK-1-binding specificity and the IL-2 biological activity. RESULTS: The FUscFv/IL-2 fusion protein effectively introduced a specific cytotoxicity of lymphokine-activated killer cells to the tumor cells and consequently suppressed the tumor growth in a SCID mouse xenograft model. CONCLUSION: This approach may be used for in vivo administration to localize IL-2 to tumor tissues, enhancing the immune response to human MK-1-expressing tumors while reducing systemic side-effects.

Targeted sonodynamic therapy of cancer using a photosensitizer conjugated with antibody against carcinoembryonic antigen.

The goal of this study was to develop a strategy for the selective destruction of cancer cells by ultrasonic irradiation in the presence of an antibody-conjugated photosensitizer. To this end, a photoimmunoconjugate (PIC) was prepared between ATX-70, a photosensitizer of a gallium-porphyrin analogue, and F11-39, a high affinity monoclonal antibody (MAb) against carcinoembryonic antigen (CEA), which is often overexpressed in various carcinoma cells. This conjugate, designated F39/ATX-70, retained immunoreactivity against purified CEA and CEA-expressing cells as determined by enzyme-linked immunosorbent assay, flow cytometry and immunofluorescence microscopic analysis. The cytotoxicity of F39/ATX-70 against CEA-expressing human gastric carcinoma cells in vitro was found to be greater than that of ATX-70 when applied in combination with ultrasound irradiation. When in vivo anti-tumor effects in a mouse xenograft model were assessed, intravenous administration of F39/ATX-70 followed by ultrasonic irradiation produced a marked growth inhibition of tumor compared with irradiation alone or irradiation after administration of ATX-70. These results suggest that the PIC between anti-CEA MAb and ATX-70 may have applications in sonodynamic therapy where destruction of CEA-expressing tumor is required.

T-cell immunotherapy for human MK-1-expressing tumors using a fusion protein of the superantigen SEA and anti-MK-1 scFv antibody.

BACKGROUND: The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we constructed a recombinant fusion protein of SEA and the single-chain variable fragment (scFv) of the FU-MK-1 antibody, which recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas. MATERIALS AND METHODS: We employed recombinant DNA techniques to fuse recombinant mutant SEA to an scFv antibody derived from FU-MK-1 and the resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system, purified with a metal-affinity column, and characterized for its MK-1-binding specificity and its antitumor activity. RESULTS: The SEA/FUscFv fusion protein retained the reactivity with MK-1-expressing tumor cells, introduced a specific cytotoxicity of lymphokine-activated killer T-cells to the tumor cells, and consequently suppressed the tumor growth in a SCID mouse xenograft model. CONCLUSION: This genetically engineered SEA/FUscFv fusion protein may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

Significance of tumor-associated antigens in the diagnosis and therapy of cancer: an overview.

An enormous effort using a wide variety of approaches has been undertaken over the last three decades to transform both basic and clinical research into improved diagnoses and therapies of cancer. This brief overview summarizes the significance of tumor-associated antigens (TAAs) in the diagnosis and therapy of cancer. Current data suggest that immunotherapy and gene therapy using antibody-recognized TAAs as their targets are promising, whereas those using T cell-recognized peptide epitopes of TAAs as their targets remain controversial regarding their efficacy, mainly due to general losses of HLA molecules in tumor cells.