Objectives:
Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is the most
important human immune checkpoint that modulates T cells activity and
brings about immune-homeostasis. Accordingly, checkpoint inhibitor
cancer therapy has been approved as a growing method to block
over-expressed immune checkpoints, such as CTLA-4 receptors. Considering
the competitive characteristics of single-domain antibodies with
monoclonal antibodies, we tried to develop a camelid Nanobody against
human CTLA-4.
Materials and methods:
We have constructed the VHH gene library by using immunized-camel
peripheral blood mononuclear cells and carrying out the Nested-PCR
technique. VHH-library was screened by phage display technique and
specific nanobodies against CTLA-4 protein were selected and amplified
with bio-panning steps. Stronger binders were screened by Periplasmic
Extract-ELISA, followed by estimating the complexity of the library.
Specific anti-CTLA-4 Nanobody and 3hCTL55, with longer CDR3 and a higher
binding rate, were selected for more assays.
Results:
Results revealed the existence of two different clones in the library with 108
binders. In comparison with seven different antigens, using the ELISA
technique confirmed the specificity of Nanobody 3hCTL55 against human
CTLA-4 antigen. We calculated Nanobody 3hCTL55 affinity for human CTLA-4
antigen at 50×10-9 M, approximately. Performing western blot
and Flow-cytometry techniques showed that Nanobody 3hCTL55 was able to
specifically detect and attach both commercial human CTLA-4 protein and
human CTLA-4 antigen on the cell surface and in the cell lysate.
Conclusion:
Taken together, this developed camelid-specific anti-CTLA-4
Nanobody 3hCTL55, selected from a high-quality immune library by phage
display technique, may be effective for further study about cancer
diagnosis and cancer-therapy purposes.