| In pursuit of recombinant antibodies for tumor immunotherapy, two independent projects were undertaken. In the first, H60 fusion proteins were constructed, characterized, and tested in two murine immunotherapeutic models. H60 is a murine minor histocompatibility antigen that binds NKG2D and activates NK cells. Two H60 fusion proteins were generated: the tumor-targeting H60/TNT-3, and the non-tumor-targeting H60-Fc control. The Ka of H60/TNT-3 (2.43 x 109 M-1) was nearly identical to that of the parental mAb (2.22 x 109 M-1). In vitro, H60 fusion proteins bound and activated murine NK cells, eliciting IFN-gamma production in a higher percentage of cells than the activating NKG2D Ab A10. In vivo, H60/TNT-3 effectively targeted tumor tissue, with nearly 2% injected dose per gram of tumor retained after 48 h. H60 fusion proteins were tested for anti-tumor efficacy in CT26/BALB/c and in Lewis Lung/C57BL/6 tumor models. Tumor volume reduction was observed in both models (53% and 52%, respectively) relative to untreated control mice. Further, Lewis Lung carcinoma-bearing mice treated with H60/TNT-3 experienced a statistically significant survival advantage. Taken together, these data characterize a new immunotherapeutic MAb with anti-tumor efficacy that prolonged overall survival in a resistant solid tumor model. For the second project, a novel autoimmune phage display library was generated with the intent of producing new Tumor Necrosis Treatment (TNT) antibodies, which target the necrotic regions of solid tumors. This autoimmune library consisted of variable immunoglobulin genes obtained from two strains of autoimmune mice (NZBWF1 and MRL/MpJ-Tnfrsf6lpr) pre-immunized with nuclei. The resultant library was panned in vivo by intravenous injection into CT26 tumor-bearing BALB/c mice. After three rounds, 10 7phage particles were retrieved from experimental tumors. By contrast, in vivo panning with wild-type M13 phage yielded no output. From the large pool of autoimmune library phage obtained, 61 clones were analyzed. The Round 3 output pool was characterized by a high percentage of unique scFv sequences (39%). A flow cytometric assay was developed to measure binding of individual phage clones to nuclear antigens in permeabilized cells, and may be used in the future for high-throughput analysis of Round 3 output phage clones. |
