Generation of polyclonal antibodies is relatively simple and cheap as the use of animals (such as chicken, horses, goats, and rabbits) enables the recovery of large quantity of antibodies from serum or egg Yolk (Yakhkeshi et al., 2022 ). Polyclonal antibodies arising from diverse B-cell clones constitute a heterogeneous mixture that manifests varying binding affinities. In contrast, monoclonal antibodies are homogeneous population originated from a single B-cell clone, resulting in well-defined binding specificities and affinities. However, at some point a fresh batch will be sought as the original stock diminishes, which inevitably leads to a batch-to-batch variation in terms of the specificity, sensitivity, and reproducibility (Bradbury & Plückthun, 2015 ). This might include differences in antibody reactivity and titre, and thus polyclonal reagents often lack reproducibility. In comparison, the continuous culture of B cell hybridomas overs a reproducible and potentially inexhaustible supply of antibody with exquisite specificity. Consequently, monoclonal antibodies enable the development of standardised and secure immunoassay system.
Genetically engineered antibody fragments are alternative to full-length antibody in diagnostics and therapeutics for a variety of diseases due to their various advantages (Holliger & Hudson, 2005 ). Firstly, their small size and simpler structure make them ideal for large-scale production in eukaryotic systems, such as mammalian and insect cells. The antibody fragments have known sequences and are reproducible, verifiable, and manufacturable. The single-chain variable fragment (scFv) is a successful example of a genetically engineered antibody fragment. As the variable light chain and variable heavy chain coding sequences are genetically linked in a single transcript, there is no need to balance the expression of the light chain and heavy chain. Engineered single domain antibodies (VHH) enable the rapid generation of antibody fragments at higher yields and lower cost compared to full-size monoclonal antibodies (mAbs) that typically require mammalian expression systems. Their small size also facilitates tissue penetration and access to cryptic epitopes, making them particularly useful for tumour penetration in cancer immunotherapy (Zinn et al., 2023 ). Moreover, the lack of an Fc region removes the risk of bystander immune cell activation and antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC), allowing the molecule to bind its target without activating the host’s immune system (Gravina et al., 2023 ). However, the absence of an Fc region is a double-edged sword. The disadvantage is that it not only reduces thermostability and enhances propensity for aggregation, thereby increasing the risk of immunogenicity, but also shortens half-life due to a lack of FcRn-mediated recycling (Park et al., 2016 ). This can lead to the need for higher and more frequent dosing. Fusion of the scFv to albumin or polyethylene glycol (PEG) can be used to improve half-life (Albrecht et al., 2004 ;Vazquez-Lombardi et al., 2015 ). However, such fusions can offset the advantages that an scFv holds over a mAb due to cost and the increase in size (Bradbury & Plückthun, 2015 ).
The fourth generation of antibody engineering: antibody mimetics is not generated by the body immune system naturally but share a common feature with natural antibody: they shared characteristic of complementary shape at the binding site with the antigen. Similar to enzyme and substrate, the binding interactions between antibody mimetics and antigens can be explained by three models, including the classical lock and key theory, the induced fit mode, and the conformational selection model. Typically, the classical lock and key theory reflects the initial recognition and specifically between antibody mimetics and antigens. The induced fit model suggests that both antibody mimetics and antigens undergo conformational changes upon binding, in which the dynamic interaction allows the active sites of antibody mimetics to adapt to their shape, optimizing the fit with the antigen. Additionally, the conformational selection model proposes a conformational change occurs prior to the binding of antigens, in which the antigen seems to select and stabilize a higher-energy conformation of an antibody mimetic for binding. Of note, these models are not mutually exclusive, but rather represent different aspects of interactions between antibody mimetics and antigens. Antibody mimetics represent a class of engineered molecules mimic the functions and properties of natural antibodies while offering advantages such as enhanced stability, smaller size, improved production yields. These mimetics can manifest in various forms, like smaller antibody fragments synthesized based on the functional regions of antibody fragments such as scFv or Fab. It is possible to design novel proteins with binding properties through site-directed mutagenesis and random mutagenesis. We summarized and compared the features of all the four generations of in terms of stability, specificity, affinity, and so forth (Table 1).
Now it may be the right time to review and analyse the field of antibody mimetics retrospectively and prospectively. Given that scientometric analysis can quantitively evaluate and investigate on all aspects of the literature at different development stages (Mooghali et al., 2011 ), this article aims to provide a vigorous roadmap for antibody mimetics research through this method to identify the major players and their cooperation networks, including countries, academic groups, and individuals; to analyse research status and hotspots, especially the key study findings; to discuss the potentially valuable research directions.
Table 1 Antibody production & diversified antibody generation platforms