First pan-specific vNAR against human TGF-β as a potential therapeutic application: in silico modeling assessment

Abstract

Immunotherapies based on antibody fragments have been developed and applied to human diseases, describing novel antibody formats. The vNAR domains have a potential therapeutic use related to their unique properties. This work used a non-immunized Heterodontus francisci shark library to obtain a vNAR with recognition of TGF-β isoforms. The isolated vNAR T1 selected by phage display demonstrated binding of the vNAR T1 to TGF-β isoforms (-β1, -β2, -β3) by direct ELISA assay. These results are supported by using for the first time the Single-Cycle kinetics (SCK) method for Surface plasmon resonance (SPR) analysis for a vNAR. Also, the vNAR T1 shows an equilibrium dissociation constant (K_D) of 9.61 × 10^-8 M against rhTGF-β1. Furthermore, the molecular docking analysis revealed that the vNAR T1 interacts with amino acid residues of TGF-β1, which are essential for interaction with type I and II TGF-β receptors. The vNAR T1 is the first pan-specific shark domain reported against the three hTGF-β isoforms and a potential alternative to overcome the challenges related to the modulation of TGF-β levels implicated in several human diseases such as fibrosis, cancer, and COVID-19.

Figure 1

Isolation of anti-TGF-β vNAR. (a) Panning rounds with naïve library against rhTGF-β1, (b) Analysis of the periplasmic expression of vNAR domains, (c) Recognition ELISA assay of vNAR domains periplasmic extracts against rhTGF-β1. Error bars represent standard deviation (s. d.), n = 3. ***P < 0.001, **P < 0.01, and *P < 0.05.

Figure 2

Nucleotide and aminoacidic sequences of the vNAR T1. (a) Nucleotide and aminoacidic sequences of vNAR T1. (b) Multiple Sequence Alignment (MSA) of vNAR T1 domain with other vNAR sequences (GenBank AF336089, AF336087; AF336088; AY069988; AF336094), showing a long CDR3 (24 aa) of the vNAR T1 with recognition capacity against rhTGF-β. The CDR1 region is in an orange box. The CDR3 region is in a red box. The canonical cysteine residues (amino acids: 22 and 83) in FR1 and FR3 regions (highlighted in blue). The non-canonical cysteine residues in CDR1 and CDR3 regions (highlighted in yellow). (c) Structural analysis of vNAR T1 shows that the CDR3 region acts as a hairpin with the binding capacity of TGF-β isoforms (CDR3 region in a red box). (d) Ramachandran plot of the model shows 99 residues (96.1%) in favored regions and 4 residues (3.9%) in additional regions.

Figure 3

SDS-PAGE and western blot analysis for His-tagged vNAR T1 expression and binding to immobilized rhTGF-β isoforms by ELISA (a) 12% SDS-PAGE. MWM: Molecular weight marker, NR: Non-retained, W1-2: Wash solutions, E0-5: Elution fractions 0–5. (b) Western blotting. MWM: Molecular weight marker, E0-3: elution fractions, C + : Positive control (irrelevant non-related protein with a six His tag), (c) vNAR T1 binding to immobilized rhTGF-β isoforms by ELISA. Error bars represent standard deviation (s. d.). n = 3. ***P < 0.001, **P < 0.01, and *P < 0.05, n = 3. The purified his-tagged vNAT T1 recognizes three human TGF-β isoforms compared to 3% BSA (*** P < 0.001).

Figure 4

Surface Plasmon Resonance assay (SPR). (a) Schematic representation of SCK method. (b) Representation of Single-cycle kinetics (SCK) experiments of vNAR T1/rhTGF-β1. Sensorgram (blue dotted curve) of the response (resonance units, RU) versus time of the SCK by injecting five increasing concentrations (87.5 nM to 1,400 nM) of rhTGF-β1 over the vNAR T1 His-tag + Anti-His Antibody. The result represents the mean of three independent experiments.

Figure 5

Structural analysis of vNAR T1 coupled to human TGF-β isoforms and competition binding assay. (a) vNAR T1 (blue) with TGF-β1 (yellow and green). The grey boxes show a detailed view of the interfacing residues (polar contacts as orange dash lines). (b) vNAR T1 (blue) interaction with TGF-β2 isoform (yellow and grey). (c) vNAR T1 (blue) interaction with TGF-β3 isoform (yellow and orange). All TGF-β isoform shows as a homodimeric soluble protein. (d) Sequence alignment of each TGF-β isoform, the interface residues of TGF-β1 interacting with the natural receptor type 2 (TβRII) are highlighted in blue, and the amino acids recognized for type 1 receptor (TβRI) were shown in green both (from the complex entry in PDB ID 3KFD). In red color are highlighted the residues of TGF-β amino acids recognized by the vNAR T1 domain.

Figure 6

In silico competition binding assay of vNAR T1 and TβRII against TGF-β. The main amino acids of the complexes vNAR T1/TGF-β1 and TβRII/TGF-β1 are focused into de boxes and represented with amino acid abbreviations and numbers.