Screening and antitumor effect of an anti-CTLA-4 nanobody

Cytotoxic T‑lymphocyte antigen‑4 (CTLA‑4) is a critical negative regulator of immune responses. CTLA‑4 is rapidly upregulated following T‑cell activation, and then binds to B7 molecules with a higher affinity than CD28. CTLA‑4 may abolish the initiation of the responses of T cells by raising the threshold of signals required for full activation of T cells, and it also may terminate ongoing T-cell responses. This regulatory role has led to the development of monoclonal antibodies (mAbs) designed to block CTLA‑4 activity for enhancing immune responses against cancer. mAbs have several disadvantages including high production cost and unstable behavior. Nanobodies (Nbs) are single‑domain antigen‑binding fragments derived from the camelid heavy‑chain antibodies, which are highly attractive in cancer immunotherapy due to their small size, high specificity, and stability. We selected CTLA‑4‑specific Nbs from a high quality dromedary camel immune library by phage display technology. Four positive colonies were sequenced and classified based on the amino acids sequences in the CDR3 region. These Nbs recognized unique epitopes on CTLA‑4 and displayed high binding rates when used on PHA‑stimulated human T cells. Treatment of B16 melanoma‑bearing C57BL/6 mice with anti‑CTLA‑4 nanobody 16 (Nb16) delayed melanoma growth and prolonged the survival time of mice. These data indicate that anti‑CTLA‑4 Nbs selected from a high quality phage display library may be effective for the treatment of patients with tumors.