Development by Genetic Immunization of Monovalent Antibodies Against Human Vasoactive Intestinal Peptide Receptor 1 (VPAC1), New Innovative, and Versatile Tools to Study VPAC1 Receptor Function

Abstract

Multi-membrane spanning proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are extremely difficult to purify as native proteins. Consequently, the generation of antibodies that recognize the native conformation can be challenging. By combining genetic immunization, phage display, and biopanning, we identified a panel of monovalent antibodies (nanobodies) targeting the vasoactive intestinal peptide receptor 1 (VPAC1) receptor. The nine unique nanobodies that were classified into four different families based on their CDR3 amino acid sequence and length, were highly specific for the human receptor and bind VPAC1 with moderate affinity. They all recognize a similar epitope localized in the extracellular N-terminal domain of the receptor and distinct from the orthosteric binding site. In agreement with binding studies, which showed that the nanobodies did not interfere with VIP binding, all nanobodies were devoid of any functional properties. However, we observed that the binding of two nanobodies was slightly increased in the presence of VPAC1 agonists [vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27)], but decreased in the presence of VPAC1 antagonist. As no evidence of allosteric activity was seen in VIP binding studies nor in functional assays, it is, therefore, possible that the two nanobodies may behave as very weak allosteric modulators of VPAC1, detectable only in some sensitive settings, but not in others. We demonstrated that the fluorescently labeled nanobodies detect VPAC1 on the surface of human leukocytes as efficiently as a reference mouse monoclonal antibody. We also developed a protocol allowing efficient detection of VPAC1 by immunohistochemistry in paraffin-embedded human gastrointestinal tissue sections. Thus, these nanobodies constitute new original tools to further investigate the role of VPAC1 in physiological and pathological conditions.

Keywords: DNA immunization; G protein-coupled receptor; llama; monovalent antibodies; vasoactive intestinal peptide; vasoactive intestinal peptide receptor 1; vasoactive intestinal polypeptide receptor.

Figure 1

Assessment of nanobody specificity. The specificity of the nanobodies was investigated by FACS using human embryonic kidney cells-297T cells transiently transfected with empty plasmid (dotted lines) or plasmids encoding human VPAC1, rat VPAC1 or human VPAC2 (filled histograms). The cells were incubated with a reference mouse monoclonal antibodies (mAb) for each receptor (bottom panels) or DyLight 650-conjugated nanobodies. Bound mAbs was detected with an APC-conjugated secondary antibody. The data are representative of three independent experiments.

Figure 2

Nanobodies competition binding experiments. Binding of DyLight 650-conjugated CA7277 (A) and C7281 (B) on CHO cells expressing VPAC1 in presence of increasing concentrations of CA7277 (filled circles), CA7281 (filled squares), CA7293 (open circles), or CA7295 (open squares). The data represent the means ± SEM of three independent experiments performed in duplicate.

Figure 3

Vasoactive intestinal polypeptide (VIP) binding experiments. (A) Competition binding on CHO cells expressing VPAC1 using [¹²⁵I]-VIP as tracer in presence of increasing concentrations of CA7277 (filled circles), CA7281 (filled squares), CA7293 (open circles), or CA7295 (open squares). (B) Kinetic studies of the dissociation of [¹²⁵I]-VIP from CHO cells expressing VPAC1 (after 30 min of association at 23°C), following addition of 1 µM VIP (filled diamonds), 3 µM CA7277 (filled circles), CA7281 (filled squares), CA7293 (open circles), CA7295 (open squares), or buffer (open diamonds). The results are the means ± SEM of three independent experiments performed in duplicate.

Figure 4

Effect of VPAC1 ligands on nanobodies binding. Binding of DyLight 650-conjugated CA7277 (A) and C7281 (B) on CHO cells expressing VPAC1 in presence of increasing concentrations of VIP (filled circles) or VPAC1 antagonist (filled squares). The results are the means ± SEM of three independent experiments performed in duplicate.

Figure 5

Effect of nanobodies on cAMP accumulation. (A) Cyclic AMP accumulation in the presence of VIP (filled circles), CA7277 (open circles), or CA7281 (open squares) in CHO cells expressing VPAC1 (left panel). Effect of CA7277 (B) or CA7281 (C) at 100 or 300 nM on cAMP accumulation promoted by increasing concentrations of VIP in the same cells. The results are the means ± SEM of three independent experiments performed in duplicate.

Figure 6

Effect of nanobodies on pituitary adenylate cyclase-activating polypeptide (PACAP)-bound VPAC1. Binding of DyLight 650-conjugated CA7277 (A) and C7281 (B) on CHO cells expressing VPAC1 in presence of increasing concentrations of PACAP-27 (filled circles) or PACAP-38 (filled squares). Effect of CA7277 (C,E) or CA7281 (D,F) at 100 or 300 nM on cAMP accumulation promoted by increasing concentrations of PACAP-27 (C,D) or PACAP-38 (E,F) in CHO cells expressing VPAC1. The results are the means ± SEM of three independent experiments performed in duplicate.

Figure 7

Immunodetection of VPAC1 on human primary cells. Cells were stained for leukocyte markers (CD3, CD4, CD8, CD14, CD11b, and CD206) and VPAC1 labeling was investigated on human T cells (first column), CD4⁺ T cells (second column), CD8⁺ T cells (third column), monocytes (fourth column), and monocyte-derived macrophages (fifth column), using a reference anti-VPAC1 mouse monoclonal (mAb) and DyLight 650-conjugated nanobodies. The background signal (dotted lines) was evaluated using a control isotype labeled with APC, or a DyLight 650-conjugated irrelevant nanobody. The data are representative of three independent experiments.

Figure 8

VPAC1 immunohistochemical staining in the normal human gastrointestinal tract. Sections of human stomach (left panels), small intestine (middle panels), and colon (right panels) were dewaxed, microwaved in citrate buffer, and incubated with 300 nM CA7281 (top panels). Bound nanobodies were detected using the peroxidase-antiperoxidase (PAP) method and sections were counterstained with hematoxylin and eosine. Bottom panels show the results obtained on adjacent sections treated similarly, but omitting the nanobody. Pictures were taken on a Zeiss Axioplan two microscope (original magnification 20×).