Cancer Immunol Immunother (1994) 39:391 - 396 ancer mmunology mmunotherapy 9 Springer-Verlag 1994 Unprimed CD4+ and CD8 + T cells can be rapidly activated by a CD3 • CD19 bispecific antibody to proliferate and become cytotoxic Inez-Anne Haagen 1, Wim B. M. de Lau 1 , Bert J. E. G. Bast1, Anette J. G. Geerarsl, Mike R. Clark3, Bert C. de Gast2 I Department of Immunology, University HospitaI of Utrecht, The Netherlands 2 Department of Haematology, University Hospital of Utrecht, The Netherlands 3 Department of Pathology, University of Cambridge, UK Received: 6 June 1994/Accepted: 11 August 1994 Abstract. We previously reported that a CD3xCD19 bi- specific antibody (bsAb) can induce efficient killing of tumour cells by preactivated T cells isolated from patients with B cell malignancy. For future intravenous application we investigated whether resting T cells from peripheral blood can be stimulated to proliferate and become cyto- toxic with the CD3 x CD19 bsAb alone. Indeed peripheral blood mononuclear cells, isolated from healthy donors or patients with B cell malignancy, started to proliferate within 1 day in response to CD3xCD19 bsAb. Within the same time span cytotoxic activity against CD19-positive tumour cells was already detectable. Maintenance of cy- totoxic activity was seen during 3 days of culture but op- timal lysis of the target cells then required fresh CD3 x CD19 bsAb in the cytotoxicity assay. Essentially the same results for proliferation and cytotoxicity were found when separated CD4-positive and CD8-positive T cells were activated by the bsAb in the presence of autologous monocytes. These results may be relevant for the in vivo application of the bsAb when used as immunotherapy in patients with B cell malignancy. Key words: Immunotherapy - Bispecific monoclonal antibodies - T cell activation - Cytotoxicity Introduction Bispecific antibodies (bsAb) combining reactivity against the T cell receptor (TCR)/CD3 complex and a tumour cell This work was supported by grant IKMN 90-10 from the Dutch Cancer Society. M.C. was supported by a grant from the UK Medical Research Council Correspondence to: I.-A. Haagen, Department of Immunology (F03.821), University Hospital Utrecht, Postbox 85500, 3508 GA Utrecht, The Netherlands antigen can induce T cells to lyse these tumour cells; this is known as targeted cytotoxicity. Most T cells from the pe- ripheral blood (PBL-T) have little or no cytotoxic activity. Therefore, PBL-T have been activated ex vivo with mito- gens or CD3 mAb in combination with interleukin-2 (IL-2), loaded with bsAb and used for loco-regional administration in patients with solid tumours (reviewed in [9, 10, 25]). For haematological turnouts, including lymphoma, loco-regio- hal administration is not sensible because these tumours are generally disseminated; intravenous administration of bsAb seems to be more logical. Apart from logistic problems, ex vivo activation and expansion of T cells results here in an unfavourable recirculation pattern of the activated T cells [23]. Naive T cells traffic preferentially through lymphoid tissues and low numbers are found in non-lymphoid tissues whereas memory T cells and activated T cells or T cell clones have a strong preference for non-lymphoid tissues, especially the lungs and the liver (reviewed in reference [17]). The problem of an altered recirculation pattern may be avoided, when the bsAb by itself can activate T cells in vivo in lymphoid tissues. Cross-linking of the CD3 com- plex by the bsAb may occur locally when the bsAb-coated T cell comes across target tumour cells expressing the second antigen recognized by the bsAb. Transduction sig- nals through the TCR/CD3 complex may then lead to functional responses including cytotoxicity and production of cytokines [27]. In this study we describe the activation, with CD3xCD19 bsAb only, of peripheral blood mononuclear cells (PBMC) isolated from healthy donors and patients with non-Hodgkin's lymphoma (NHL) in remission. In all PBMC cultures the development of cytotoxic T cells in response to CD3 x CD 19 bsAb activation was seen within 2 days of stimulation. This held for both the CD4 + and the CD8 § T cell subset. Materials and methods Bispecific monoclonal antibody: SHR-1. The SHR-1 is a fusion pro- duct between the YTH12.5 and the MG1CD19 cell lines. YTH12.5 is a