A novel NKG2A alpaca nanobody of immune checkpoint blockade for the treatment of malignant melanoma

Xiang Guo^1,2,3Congfang Guo⁴Dongxiao Li³Yuting Bai⁵Mureed Abbas^6,7Ruiwen Fan³Yiyan Zhao^1,2*

• ¹Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, China
• ²Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
• ³College of Animal Science, Shanxi Agricultural University, Taigu, Shanxi, China
• ⁴Tianjin First Central Hospital, Tianjin, China
• ⁵Department of Energy Chemistry and Materials Engineering, Shanxi Institute of Energy, Jinzhong, China
• ⁶Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi Province, China
• ⁷College of Life Science, Shanxi University, Taiyuan, China

Alpaca is a camelidae animal. Antibodies produced by alpaca immunization are called nano-antibodies. Compared with traditional antibodies, nanoantibodies have the characteristics of small molecular weight, stable structure, high homology with human antibodies, and suitable for prokaryotic expression. Malignant Melanoma (MM) is a significant malignant tumor affecting both humans and animals. It commonly develops in the mucous membranes of the skin, nose, mouth, anus and digestive tract as well as in the choroid of the eyes. Melanoma is the most aggressive and lethal form of skin cancer. There are several factors that contribute to melanocyte carcinogenic, including UV radiation, endocrine disorders, viral infections, immune deficiencies and chemical carcinogens ect. While the etiology of melanoma is not yet clear. At present, surgical resection is the main treatment for MM, but the prognosis is poor. Targeted therapy and immune checkpoint inhibitors (ICIs) are increasingly utilized in the clinical treatment of melanoma.NKG2A protein is an inhibitory receptor protein on the surface of CD8 + T cells and NK cell membranes. HLA-E ligands on the surface of malignant melanoma cells could facilitate immune escape of tumor cells by specifically recognizing the NKG2A receptor complex on the surface of immune cells. This recognition mechanism enables tumor cells to evade detection by the immune system through immune-suppressive processes. Immunosuppressive antibody drugs activate immune cells to target and kill tumor cells by blocking this recognition mechanism. Thus, NKG2A is a novel target for such therapeutic interventions. In this study, take advantage of the alpaca nanoantibody, and a monoclonal nanobody strain expressing NKG2A protein with high affinity was obtained from the phage library of melanoma. NKG2A nanobody with high affinity was obtained through induced expression and protein purification, with potential applications in the detection and treatment of MM.