The conventional hybridoma method of antibody production is time consuming and laborious. Previously, we have produced some monoclonal antibodies against MIC [17
]. Anticancer drugs conjugated directly to antibodies are not efficient, and only a limited number of drug molecules could be conjugated leading to low drug carrier. In order to produce MICA-targeted anticancer drug, monoclonal antibodies against MICA were cloned and displayed on filamentous bacteriophages. Unlike drug conjugated to an antibody, a large amount of anticancer drug can be easily conjugated by chemical linking to pVIII which exists 2,700 copies as major-phage-coated proteins per a phage particle [21
]. Anticancer-drug-conjugated bacteriophages have been used successfully with a potentiator factor of over 1,000 compared to free drug [22
]. The studies of phages as targeted drug carriers were mostly in vitro
. The in vivo
study was only in a murine model [23
]. Immunogenicity of bacteriophages in drug administration would be challenging and required further in vivo
studies, especially in human.
The role of NKG2D receptor and ligands in immune responses against cancer is well established and has been exploited as approaches for cancer immunotherapy. These include the induction of anti-MICA to stimulate antitumor cytotoxicity [24
], therapeutic DNA-based vaccine of NKG2D ligands and tumor antigens [25
], and the generation of T cells with chimeric NKG2D receptors directly activated by ligand engagement [26
]. However, the approaches employing drug conjugated to anti-NKG2D ligands have not been reported.
Apparently, the original anti-MICA displayed phages had less activities to detect MICA compared to monoclonal antibodies (). It has been shown that mutations of HCDR3 of antibodies could allow antibodies to improve binding activity and specificity [21
]. Thus, we performed in vitro
affinity maturation by randomly mutating CDR3 which is one of the antigen-binding domains and selected for phages with higher activities by several rounds of biopanning. According to our data, at least seven rounds of selections would be needed for maximal enhancement. Normally, in other studies the panning was performed for only 3–5 rounds [18
] resulting in a few positive antibody clones. Additional rounds of panning of up to ten rounds did not increase the collective OD. Among 61 individual clones of mutants, we obtained only fives clones (8.19%) that had high binding activities. Two mutant clones with highest activities were characterized by flow cytometry () and sequencing (data not show). These phage clones were able to recognize MICA in a native form according to positive results obtained by both indirect ELISA and flow cytometry. To further characterize the high activities clones, competitive ELISA will be performed as well as testing against allelic MICA proteins to determine whether these clones have any limitation in detecting diverse MICA alleles.
In conclusion, phage display technology is an effective method to generate and isolate high affinity monoclonal antibodies against specific antigens. Phages displaying anti-MICA with high binding activities have been established. By conjugation with anticancer drug such as doxorubicin or 5-FU, these reagents would have high potential to develop targeted therapy against cancer cells expressing MICA proteins.