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

Abstract

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.

Novel Delivery Systems for Checkpoint Inhibitors

“…With the recent approval of the first therapeutic nanobody (targeting von Willebrand factor) for treatment of acquired thrombotic thrombocytopenic purpura [115], there is much to look forward to in the successful application of nanobodies in cancer immunotherapies. While design and efficacy of nanobodies targeting checkpoint molecules have been evaluated [116,117,118], studies evaluating the feasibility and efficacy of targeted delivery of nanobodies are scarce.…”

Highly sensitive detection of CTLA‑4‑positive T‑cell subgroups based on nanobody and fluorescent carbon quantum dots

“…Previously, Nb36 protein was obtained from a high quality dromedary camel immune library by phage display technology and expression in E. coli WK6 electrocompetent cells. After further purification, the Nb36 protein solution was obtained (10). First, Nb36 protein solution was dissolved in the PBS (to a final concentration of 1 mg/ml).…”

“…Compared with conventional antibodies, Nbs possess several inherent characteristics, including high specificity, high physiochemical stability, lack of immunogenicity, high yield and low cost, which make them suitable for immune-targeted diagnosis and treatment of cancer (9). In our previous study, a CTLA-4-specific Nb (Nb36) was screened (10). Nb36 may recognize unique CTLA-4 epitopes and effectively bind with CTLA-4 + T cells.…”

Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics

“…Various studies have created nanobody ICIs for PD-L1 ( 36 , 77 – 81 ), enhancing anti-tumor efficacy when combined with its mAb counterpart, avelumab in vivo ( 36 ). Anti-CTLA-4 nanobodies have also demonstrated anti-tumor effects ( 39 , 82 ); however, Ingram et al ( 39 ) study suggest that an Fc domain may be needed for clinically-relevant potency. Homayouni et al ( 83 ) developed the first nanobody targeting T-cell immunoglobulin and mucin domain 3 (TIM-3), demonstrating anti-proliferative effects in vitro .…”