1. Introduction
Antibodies, ~150 kDa glycoproteins, consist of two light
and two heavy polypeptide chains with six CDRs for specific binding to
their targets [1]. However, their therapeutic use is limited due to
the low tissue penetration. Recently, advances in the structural
engineering of proteins led to the construction of many small-size
proteins (3-20 kDa) with the ability to bind to epitopes recognized by
mono antibodies [2]. Among all the engineered and recombinant
proteins, single chain variable fragments (scFvs) composed of VH and VL,
with the same antigen-binding specificity can retain the affinity of a
whole antibody molecule [3]. Low-molecular weight, high rate of
excretion, good tissue penetration, and low costs for production of
these antibody mimetics converted them into a promising alternative for
targeted therapies [4]. Currently, amino acid conversion on the
surface area of non-immunoglobulin scaffolds resulted in the generation
of various small antibody mimetics [4]. However, length,
conformation, and the conserved amino acid residues of the hypervariable
CDRs loop play an essential role in native binding site definition
[5]. On the other hand, 30 kDa fragments of antibody mimetics
developed by the antibody fragmentation technique restricted their
simple chemical synthesis [4]. Using the CDRs grafting strategy,
protein scaffolds with variable loops that mimic the conformation of
CDRs can be extracted to generate small scaffolds with improved
affinity. Nicaise et al. developed some protein scaffolds against
lysozyme which harbored CDRs from a specific antibody. However, designed
scaffolds showed various affinities towards lysozyme, indicating that
selected scaffolds directly affect the affinity of grafted CDRs [6].
Therefore, for designing antibody mimetics, selecting proper protein
scaffolds and determining appropriate CDRs peptide sequences is
essential [7]. Among many introduced non-immunoglobulin scaffolds,
anticalins with a smaller size of 180 amino acids consisting one
polypeptide chain, a beta-sheet structure, and four flexible loops
without any glycosylated structure can be produced in prokaryotic hosts
[8]. Another example of protein scaffolds is the tenth domain of
human fibronectin type III (Fn3, also called Adnectin). Adnectin is a
94-residue monomeric protein with a β-sandwich fold structure and six
internal loops without disulfide bonds. This protein mimics the
interaction of CDRs with the target through three loops on the tip of
molecules [8]. The third structure selected for this study is VHH,
with small molecular weight, low aggregation, and high solubility, which
contains three variable loops [5]. This existing assortment
represents an ideal CDR acceptor repertoire for our approach. In
addition to the structure of the CDRs acceptor, the framework plays a
vital role in CDR orientation and conformation. Therefore, framework
residues should be considered in designing a protein scaffold with high
binding affinity to the target [9]. Here, we aimed to simulate the
CDRs conformation in three selected scaffolds of humanized VHH, Fn3, and
lipocalin with at least three flexible loops for grafting CDRs isolated
from the target antibody. Three CDRs peptides of H2, H3, and L3 that
play an important role in the recognition of the target were extracted
from the monoclonal antibody panitumumab that binds specifically to the
EGFR DIII. These CDRs were then grafted through loop randomization in
all these scaffolds. Apart from scFv, which carries all 6 CDRs in their
native structure, 36 anti-EGFR DIII protein candidates were created (24
candidates from lipocalin scaffold, 6 structures of Fn3, and 6
candidates of humanized VHH). Among all these structures, three of them
were selected for further analysis in molecular dynamic simulation.