pComb3 vector

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a key negative regulator of the immune response. CTLA-4 can abrogate the initiation of T cell responses by raising the signaling threshold required for full T cell activation and can also terminate ongoing T cell responses. Nanobodies (Nbs) are single-domain antigen-binding fragments derived from camel heavy chain antibodies that are extremely attractive in cancer immunotherapy due to their small size, high specificity, and stability. In this study, CTLA-4-specific Nbs were selected from a high-quality dromedary immune library via phage display technology. These Nbs recognize unique epitopes on CTLA-4 and exhibit 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) slowed melanoma growth and prolonged mouse survival. These data suggest that anti-CTLA-4 Nb selected from a high-quality phage display library may be effective in the treatment of oncology patients.

In this study, healthy camels were immunized with human CTLA-4 protein seven times, peripheral blood lymphocytes were isolated, and the VHH gene was amplified from the lymphocyte cDNA. To construct the library, PstI and NotI sites were introduced at the 5' and 3' ends of the VHH fragment, respectively. A total of 10 µg of VHH fragment and 20 µg of linearized pComb3 vector were used for ligation. Then, the recombinant plasmid was transformed into TG1 cells through 30 times of electroporation. Calculate library size by counting colonies after serial dilutions. Its size reaches 1.85 × 10⁸ colonies, which enables the acquisition of Nbs with high specificity and sequence diversity. 24 colonies were randomly selected for PCR analysis, and the PCR results showed that the library insertion rate was 95%. All these indicate that a high-quality immune phage display library has been successfully constructed, which provides a basis for subsequent screening of CTLA-4-specific Nb.

Figure 1. Construction of the VHH library. (Wan, Ruirong, et al. 2018)