The development of a bi-specific anti-CD161A x anti-tumor antibody for rat NK cell targeting

• Published in: Immunobiology (1979) 1999-02, Vol.200 (1), p.31-48
• Main Authors: Hagenaars, M, Ensink, N G, Jonges, L E, Chambers, W H, Eggermont, A M, van de Velde, C J, Fleuren, G J, Kuppen, P J
• Format: Article
• Language: English
• Subjects: Adenocarcinoma; Animals; Antibodies, Bispecific; Antigens, Neoplasm - immunology; Antigens, Surface - immunology; Apoptosis; Colonic Neoplasms; Cytotoxicity, Immunologic; Immunotherapy, Adoptive; Killer Cells, Natural - immunology; Lectins, C-Type; NK Cell Lectin-Like Receptor Subfamily B; Rats; Tumor Cells, Cultured
• ISSN: 0171-2985
• DOI: 10.1016/S0171-2985(99)80031-X

In order to improve the therapeutic efficacy of adoptive immunotherapy of cancer using IL-2-activated NK (A-NK) cells, we developed a bi-specific monoclonal antibody (BimAb) 3.2.3xCC52. One specificity of the BimAb (mAb 3.2.3) was directed against rat CD161A (NKR-P1A) which has been shown to be an activation structure on rat NK cells involved in lysis of target cells and cytokine secretion. The other specificity (mAb CC52) was directed against a tumor associated antigen on the rat colon adenocarcinoma cell line CC531. The hybridomas producing 3.2.3 and CC52 were fused, resulting in a quadroma producing the desired 3.2.3xCC52 BimAb. The hybridomas produced antibodies of different isotypes (IgG2b and IgG1 respectively) which enabled us to pre-select quadromas with a high likelihood for production of BimAb, through testing for the production of bi-isotypic antibodies. Production of functional BimAb by the selected quadromas was demonstrated in an assay showing enhanced conjugate formation between CD161A+ cells and CC531 tumor cells. We also tested the 3.2.3xCC52 BimAb for its capacity to enhance NK cell-mediated lysis of CC531 tumor cells in 4 h and 19 h 51Cr release assays; in a prolonged (2 day) tumor neutralization assay using a tetrazolium salt (MTT)-based assay; and in tests for apoptosis using Annexin V-FITC. Although this BimAb was not demonstrated to cause enhanced lysis of CC531 cells by CD161A+ effector cells in vitro, it might be a useful tool to enhance the number of NK cells at the tumor site and/or prolong contact between tumor cells and NK cells in vivo, thereby probably enhancing the therapeutic efficacy of NK cells.