Chimeric T cell receptors with specificity for tumor-associapletely humanized chimeric receptor. After transfection, the ted antigens are successfully used to target T cells to humBW431/26 scFv-CH2CH3-␥ receptor is expressed as tumor cells. The efficacy of this approach, however, is a homodimer on the surface of MD45 T cells. Co-incureduced by soluble antigen that is frequently present in bation with CEA + tumor cells specifically activates grafted high serum concentrations. To overcome this situation, we MD45 T cells indicated by IL-2 secretion and cytolytic constructed an anti-CEA chimeric receptor whose extraactivity against CEA + tumor cells. Notably, the efficacy of cellular moiety is composed of a humanized single chain receptor-mediated activation is not affected by soluble CEA antibody fragment (scFv) derived from the anti-CEA mAb up to 25 g/ml demonstrating the usefulness of this chim-BW431/26 and the CH2/CH3 constant domains of human eric receptor for specific cellular activation by membraneIgG. The intracellular moiety consists of the ␥-signaling bound CEA even in the presence of high concentrations of chain of the human Fc⑀RI receptor constituting a com-CEA, as found in patients during progression of the disease.Keywords: chimeric T cell receptor; scFv; membrane-bound CEAIn a recently developed strategy for the cellular immunotherapy of malignant diseases, the advantages of antibody-based targeting and cell-mediated cytolysis have been combined by grafting effector cells with a chimeric receptor that binds to a tumor-associated antigen. 1-5 The antigen-binding domain of the receptor is composed of a single-chain antibody fragment (scFv) derived from the heavy (VH) and light (VL) chain variable regions of a monoclonal antibody (mAb). The intracellular signalling domain is derived from the cytoplasmic part of a membrane-bound receptor involved in cellular activation. After grafting cytotoxic T cells with the chimeric receptor, an antigen-specific and MHC-unrestricted cellular immune response is directed to cells expressing the particular mAb-defined antigen (for review, Refs 6 and 7).Due to xenogeneic parts of the chimeric receptor, the efficacy in the therapy of malignant diseases, however, will be negatively influenced by the potential of a hostversus-graft reaction. In addition, soluble antigen frequently present in high concentrations in the serum of cancer patients will block the receptor of grafted effector cells thus preventing tumor cell recognition and antigendriven cellular activation upon specific receptor crosslinking. 6,8 This limitation so far restricts the chimeric receptor approach to those patients with very low amounts of soluble antigen unlikely to prevent effector cell-tumor cell interaction. Malignant tumors, however, that express carcinoembryonic antigen (CEA) are frequently accompanied by high serum concentrations of soluble CEA. 9 In this particular situation the application of the chimeric receptor-based strategy requires a receptor whose tumor cell recognition and effector cel...

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Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production of suitable detection antibodies with a precisely defined ratio of antigen-binding sites to fluorophores. Problems often arise as a consequence of inefficient and unspecific labeling which can influence antibody properties. In addition, the number of incorporated fluorophores necessitates a special normalization step for quantitation. To address these problems, we constructed different mono- and bivalent bispecific antibodies with binding site(s) for the cell surface antigens, cMET, EGFR1/HER1, ErbB2/HER2 or ErbB3/HER3 and with an additional digoxigenin-binding single-chain Fv fusion. The fluorophore Cy5 was covalently coupled to digoxigenin and quantitatively bound by the bispecific antibody. A panel of tumor cell lines was assessed under different culture conditions for absolute receptor expression levels of the indicated antigens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 + 1 bispecific antibody. The same antibodies were also used to quantify the number of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments.

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