The development of a bi-specific anti-CD 161AxAnti-tumor antibody for rat NK cell targeting

In order to improve the therapeutic efficacy of adoptive immunotherapy of c ancer using IL-2-activated NK (A-NK) cells, we developed a bi-specific mono clonal 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 a ctivation structure on rat NK cells involved in lysis of target cells and c ytokine secretion. The other specificity (mAb CC52) was directed against a tumor associated antigen on the rat colon adenocarcinoma cell line CC531. T he 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 t esting for the production of bi-isotypic antibodies. Production of function al BimAb by the selected quadromas was demonstrated in an assay showing enh anced conjugate formation between CD161A(+) cells and CC531 tumor cells. We also rested the 3.2.3xCC52 BimAb for its capacity to enhance NK cell-media ted lysis of CC531 tumor cells in 4 h and 19 h Cr-51 release assays; in a p rolonged (2 day) tumor neutralization assay using a tetrazolium salt (MTT)- based assay; and in tests for apoptosis using Annexin V-FITC. Although this BimA.b was not demonstrated to cause enhanced lysis of CC531 cells by CD16 1A(+) effector cells in vitro, it might be a useful tool to enhance the num ber of NK cells at the tumor site and/or prolong contact between tumor cell s and NK cells in vivo, thereby probably enhancing the therapeutic efficacy of NK cells.