Screening and antitumor effect of an anti‑CTLA‑4 nanobody (1)
Regulatory T-cell receptors serve as immunotherapeutic targets for enhancing activation of antitumor immune responses or reversing immunosuppressive mechanisms of tumor resistance to immune surveillance and destruction. Cytotoxic T-lymphocyte antigen-4 (CTLA-4), an essential inhibitory regulator, is responsible for the early stages of T-cell expansion that opposes the action of CD28-mediated costimulation. single domain antibodies (sdAbs; also called nanobodies; Nbs) have emerged as small (~15 kDa) antigen-binding fragments which are derived from camelid heavy-chain antibodies. They present several advantages including good solubility, thermal stability, and high expression yield.
After a healthy camel was immunized with the human CTLA-4 protein 7 times, peripheral blood lymphocytes were isolated and the variable regions of heavy-chain immunoglobulins (VHH) genes were amplified from the lymphocyte cDNA.
Specific VHHs were enriched 198-fold compared with the negative control. Twenty-four colonies were selected as positive colonies whose binding ratios were >2.
After PE-ELISA, the sequences of positive colonies were analyzed and then divided into 4 families (Nb16, Nb30, Nb36 and Nb91) according to the variety of amino acid sequences in CDR3. SDS-PAGE analysis showed that Nbs had single bands with high purity. The molecular weights of 4 Nbs were 15.30, 16.41, 16.12 and 15.48 kDa, respectively.
C57BL/6 mice were subcutaneously injected with 1×105 B16/BL6 melanoma cells in the right flank. On days 7, 10, 13 and 16, the mice were treated intraperitoneally with Nb16 (100 µg in 100 µl). Treatment of mice with Nb16 clearly delayed melanoma tumor growth and prolonged the survival time of melanoma-bearing mice
