Engineering of Nanobodies Recognizing the Human Chemokine Receptor CCR7

Abstract

The chemokine receptor CCR7 plays a pivotal role in health and disease. In particular, CCR7 controls homing of antigen-bearing dendritic cells and T cells to lymph nodes, where adaptive immune responses are initiated. However, CCR7 also guides T cells to inflamed synovium and thereby contributes to rheumatoid arthritis and promotes cancer cell migration and metastasis formation. Nanobodies have recently emerged as versatile tools to study G-protein-coupled receptor functions and are being tested in diagnostics and therapeutics. In this study, we designed a strategy to engineer novel nanobodies recognizing human CCR7. We generated a nanobody library based on a solved crystal structure of the nanobody Nb80 recognizing the β2-adrenergic receptor (β2AR) and by specifically randomizing two segments within complementarity determining region 1 (CDR1) and CDR3 of Nb80 known to interact with β2AR. We fused the nanobody library to one half of split-YFP in order to identify individual nanobody clones interacting with CCR7 fused to the other half of split-YFP using bimolecular fluorescence complementation. We present three novel nanobodies, termed Nb1, Nb5, and Nb38, that recognize human CCR7 without interfering with G-protein-coupling and downstream signaling. Moreover, we were able to follow CCR7 trafficking upon CCL19 triggering using Nb1, Nb5, and Nb38.

<p>Role of Chemokines and Chemokine Receptors in Rheumatoid Arthritis</p>

“…135 A recent study reported engineered nanobodies that target CCR7 which can be used for therapeutic and diagnostic purposes. 136 Another study indicated that both prophylactic and therapeutic treatments of CIA-humanized CCR7 mice with anti-human CCR7 monoclonal antibody led to complete resistance to CIA and arrested progression, thus highlighting that CCR7 could be a potential therapeutic target in RA. 136 One potential therapeutic target in RA is CX 3 CL1 which has been blocked in a clinical trial using a monoclonal antibody as a treatment for RA.…”

“…136 Another study indicated that both prophylactic and therapeutic treatments of CIA-humanized CCR7 mice with anti-human CCR7 monoclonal antibody led to complete resistance to CIA and arrested progression, thus highlighting that CCR7 could be a potential therapeutic target in RA. 136 One potential therapeutic target in RA is CX 3 CL1 which has been blocked in a clinical trial using a monoclonal antibody as a treatment for RA. 137 The humanized anti-CX 3 CL1 monoclonal antibody E6011 was found to be safe, well tolerated, and exhibited efficacy for 52 weeks in active RA patients who were non-responders or intolerant to MTX or TNF inhibitor therapies.…”

Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics

“…Nanobodies blocking protumor chemokines [CXCL10 ( 147 )] or fused to anti-tumor chemokines [CCL21 ( 148 )] have demonstrated functionality in microfluidic devices, but have yet to be tested in vivo . Antagonistic nanobodies for chemokine receptors such as ChemR23 ( 149 ), US28 ( 150 ), CCR7 ( 151 ), CXCR4 ( 152 – 154 ), and CXCR7 ( 154 , 155 ) have also been developed. Smolarek et al ( 156 ) developed the first nanobody inhibiting the Duffy antigen receptor for chemokines (DARC), but it has yet to be applied in tumor models.…”

Identification of Key Immune-Related Genes, Molecular Pathways and Immune Infiltration as Diagnostic and Therapeutic Candidate Targets for RA: an integrated bioinformatics-based analysis

“…It interacts with CCR7 through bimolecular uorescence complementary recognition without interfering with G protein coupling and downstream signaling. Targeted analysis of the migration and invasion process of CCR7 [74]. Chemokine C-X-C motif chemokine ligand 5 (CXCL5) has functions such as chemotaxis of neutrophils, proangiogenesis, and involvement in in ammatory responses.…”