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Single-Stranded Variable Fragment Gene Libraries Built for Phage Display: An Updated Review of Design, Selection and Application
1Programa de Pós-graduação em Biotecnologia (PPGBIOTEC), Universidade Federal do Amazonas (UFAM), Manaus, AM, Brazil
2Laboratório de Diagnóstico e Controle de Doenças Infecciosas na Amazônia (DCDIA), Instituto Leônidas e Maria Deane (ILMD/Fiocruz-Amazônia), Manaus, AM, Brazil
3Programa de Pós-Graduação em Biologia da Interação Patógeno-Hospedeiro (PPGBIO-Interação), Instituto Leônidas e Maria Deane (ILMD/Fiocruz-Amazônia), Manaus, AM, Brazil
4Programa de Pós-graduação em Imunologia Básica e Aplicada (PPGIBA), Universidade Federal do Amazonas (UFAM), Manaus, AM, Brazil
5Universidade Nilton Lins, Manaus, AM, Brazil
6Faculdade Estácio do Amazonas, Manaus, AM, Brazil
7Universidade Federal do Amazonas (UFAM), Manaus, AM, Brazil
J. Microbiol. Biotechnol. 2025. 35: e2407049
Published January 15, 2025 https://doi.org/10.4014/jmb.2407.07049
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
The researcher George Pieczenik Smith was the first to report in a practical way the use of the phage display technique in 1985, which caused positive impacts in the areas of immunology and molecular biology [1]. It is a method for manipulating the DNA of a bacteriophage to build phage libraries that are capable of encoding molecules to express them in the viral capsid [2].
Currently, this technique is the main molecular tool used to select the single-chain variable fragment (scFv). The scFv is a functional antigen-binding domain containing approximately 30 kDa, formed by the light-chain variable region (VL) and the heavy-chain variable region (VH) and joined by a peptide linker [3]. This antibody format can be rapidly constructed, expressed in different hosts and, due to its reduced size, greater stability and high specificity, has advantages in therapeutic applications and diagnostic tests [4, 5].
Previous studies have reported and evaluated the development and selection of scFvs directed to numerous targets [6–10], which were selected from different gene libraries that differ in origin, construction methods, size and diversity [11]. Constructed libraries can be immune, naïve, synthetic and semi-synthetic in nature [12]. Each approach has its advantages and limitations and, depending on the nature and subsequent use of the ligand analyzed during selection, is suitable for the most varied purposes [13]. Immune libraries require prior immunization of human or animal models with specific antigens, which results in a high affinity for the isolated ligands [14].
Naïve libraries, on the other hand, do not follow classical immunization, since they use a gene repertoire that is usually derived from B cells of donor patients who have not been immunized. However, these are capable of generating molecules with different specificities [15]. Synthetic and semi-synthetic libraries also do not require immunization and can be prepared using bioinformatics analysis to improve the affinity of the molecule, employing random combinatorial mutations in regions responsible for antigen binding [16]. Finally, from any of these possible gene repertoires, the construction of phage libraries, screening and selection of scFv that binds strongly and specifically to its target is performed [17].
Decades after of the emergence of phage display, many advances in this scientific field have already been achieved and are duly documented in a vast extension of studies. However, most reviews on the production of scFvs focus on presenting general aspects of the technique and its applications [18, 19]. Thus, there are no recent reports that describe in a compiled form the diversity, complexity and applicability of the gene libraries used in the process.
The main focus of this review is to provide an up-to-date understanding of the latest research involving scFv gene libraries, in addition to offering guidance to researchers in the field regarding the choice of the best strategy for obtaining scFvs that meet medical and industrial demands. Thus, this review presents the different categories of scFv libraries already developed in the last five years, as well as the origin of the gene repertoire, library size, selection methods, molecular strategies, quality of the selected scFvs, directed targets and the costs versus benefits of the production.
Overcoming Limitations in Recombinant Antibody Generation Using Phage Display
At the end of the nineteenth century, it was found that serum from convalescent human and equine individuals from a bacterial infection could be used to treat this disease, both in humans and animals [20]. However, prolonged use of this therapy caused adverse reactions in patients, probably due to the presence of unknown proteins [21]. In 1960, the structure of antibodies was discovered as scientists sought a better understanding of the mechanisms behind serotherapy [22]. Advances in genetic engineering contributed to improvements in the process of obtaining and purifying antibodies, culminating in the emergence of monoclonal antibodies (mAbs) in 1970 [23]. These mAbs were obtained by hybridoma technology; however, the mAbs produced in murine models when used in human patients were not well tolerated for long periods [24].
The demand for more tolerable options from the clinical and therapeutic point of view fostered several studies with bacteriophages in the 1980 [25]. Bacteriophages are viruses found in nature that have a natural tropism to infect bacterial cells [26]. All the knowledge acquired about bacteriophages helped George Smith to develop the phage display technique when he conducted experiments at the University of Missouri in 1985, research that won him the Nobel Prize in 2018 [26].
In his study, Smith observed that it was possible to insert exogenous DNA next to gene III that encodes a phage surface coat protein, creating a fusion protein expressed in the viral capsid in an accessible and functional way, without compromising the infective activity of the phage [1]. Complementary studies have proven the feasibility of isolating genes of interest from random libraries [27, 28]. Below is a timeline showing the technological advances from the discovery of serotherapy to the realization of the first selections of biotechnological molecules using phage display (Fig. 1).
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Fig. 1. Chronology of the main advances from the discovery of the effects of serotherapy to the selection of the first recombinant molecules in phages.
Compared to other methodologies used to obtain therapeutic antibodies, the main advantages of phage display include the specificity of selected clones and the diversity and size of the library that can be generated [29]. The libraries comprise millions of gene sequences, with each phage particle carrying distinct sequences [5].
All the selection is performed in vitro, which promotes the rapid isolation of binding phages in various laboratory environments, thus expanding their usefulness in emergency situations [30]. Many recent studies have described the isolation of scFvs quickly and efficiently against different targets, including in the fight against the SARS-COV-2 virus, the etiological agent responsible for causing the COVID-19 pandemic [31-34].
Although scFv selection occurs in vitro, there are increasing reports of studies that have performed in vivo selections. In these cases, the phage library is injected directly into an animal model, such as mice or rabbits, and circulating phages bind directly to their target in tissues and organs [35, 36]. Phage libraries used in phage display can be stored and preserved for long periods without compromising the capacity for infection, replication, and display of the gene of interest [37]. This high viability allows their reuse and exposure to different targets aiming at the continuous discovery of new molecular interactions [38].
Depending on the chosen molecular strategy, it is possible to join the desired gene to different viral coat proteins (pIII, pVI, pVII pVIII, pIX), which can also have their structure modified to optimize surface display [39-41]. Other elements of the technique are the vectors derived from bacteriophages belonging to the genus
The practicality of the technique has resulted in a considerable number of reviews that have sought to contextualize the general aspects and synthesize knowledge about the improvements that have been achieved [43-46]. The original articles present discoveries of new drugs and therapies [47-49]. Several studies have used bioinformatics strategies to identify and perform specific adjustments in the complementarity determining regions (CDRs) of scFvs involved in the interaction with the antigen [50-53], as well as report combinations of the selection technique with other technologies.
The combination of phage display with hybridoma technology is reported to improve the cloning and expression of the scFv using the genetic information of mammalian germ cell lines [54, 55]. The combination of these two approaches combined the speed and economy of phage display to complement hybridoma technology, thus optimizing time and resources [56].
Dong
Advances in tracking clones of interest have also been achieved. One example is the use of next generation sequencing (NGS) technology, which has been adopted to increase screening and thus identify a larger number of clones from an analyzed library. In the NGS analysis, additional numbers of screened clones are observed when compared to the conventional method, which is performed using colony polymerase chain reaction (PCR) [58]. The integration of phage display with NGS is currently considered a state-of-the-art method, and is indicated for analyzing a substantial amount of gene sequences quickly and effectively [53]. NGS analysis is routinely applied to assess the quality of a library by analyzing changes in heavy chain germline usage and assessing CDR diversity, particularly the composition and length distribution of CDR-H3 [59].
In the work of Krohn
Another important role of NGS in antibody discovery occurs during the methodological process of VH / VL pairing of scFv, which allows the creation of libraries of high diversity and specificity [61]. Choe
In addition, we emphasize that the success of the use of phage display is mainly due to its simplicity, high efficiency, in vitro nature, speed and low cost, which categorizes it as a powerful tool for selecting specific ligands. The evolution of the technique is notorious and new improvements are constantly being made, which makes this technology gain more and more prominence and it contributes significantly to the diagnosis and therapy [63].
The Construction and Expression of scFv
In general, according to the origin of the gene sequence, scFvs can be constructed and selected from immune, naïve, synthetic or semi-synthetic libraries. When the source of the antibody is of an immune or naïve nature, the construction and selection of the scFvs should include the following steps (Fig. 2A): first, mRNA is extracted from the B cells of peripheral blood of human donors, whether healthy or not, or from the blood and lymphoid organs in the case of animals [5]. Then, cDNA synthesis occurs via reverse transcriptase (RT) reactions for amplification of the variable light (VL) and variable heavy (VH) chain segments. It is possible to use specific or random primer pairs that recognize defined or conserved regions, respectively, using PCR [64].
-
Fig. 2. (A) Library construction steps (B) Screening, selection and elution of binding phages (C) scFv expression and functional evaluation against its target.
The first PCR reaction aims to separately amplify the VH and VL gene. While the second reaction combines the two gene repertoires, which are assembled containing a peptide ligand, usually composed of small and hydrophilic residues of glycine (Gly) and serine (Ser) [65]. PCR is a critical step that directly interferes with cloning, and many studies present strategies for optimizing the steps and reactions to obtain a rapid construction and isolation of scFv [66-68].
During the process of building synthetic and semi-synthetic libraries, neither immunization nor manipulation of biological samples is necessary, since the entire design of the libraries is predicted through bioinformatics analyses. Synthetic libraries are chemically synthesized using oligonucleotides and semi-synthetic libraries are generated by joining synthetic regions with natural regions. These steps are performed to increase the diversity of the CDRs in the assembly process of the VH and VL genes (Fig. 2A).
After assembling the scFv gene from a natural or artificial immune gene source, the next step is to clone it fused to a phage capsid protein gene. For the isolation of high affinity scFvs, fusion to the pIII gene is recommended, as it results in a more controlled and precise presentation [4]. Among the available vector options, the pcomb3 family of vectors are preferable for the generation and selection of antibody fragment libraries.
A competent bacterium such as
Candidates identified in the first round of selection may present low affinity due to some biases such as the propagation of transforming bacteria that received the vector with the scFv gene of interest, the reduced efficiency of infection and replication of phages in the host bacteria, in addition to competition of positive clones with large numbers of mutant clones with weak affinity to the target [73, 74]. To identify high-affinity scFvs, it is recommended to perform a gradual increase in the number of screening rounds, usually, two to four rounds are necessary. This process progressively removes non-binding phages and enriches the population of phages with a higher binding strength [56].
Other parameters that affect the efficiency of biopanning screening include the concentration of immobilized antigen, the concentration of the non-ionic detergent used in the washes, and the composition of the blocking buffer [75]. Such factors require careful methodological balance, as low-stringency screening may allow retention of false positives, while high-stringency screening may result in the removal of positive clones.
The success of the bioprospection depends on the quality and format of the antigen presentation, which is crucial in the isolation of scFvs, and requires the establishment of different biopanning strategies [38, 76]. A recent study by Kamstrup
At the end of the selection rounds, although the selected molecules are not monoclonal in origin, they will have high affinity and binding strength comparable to or superior to conventional mAbs [56]. The final steps consist of sequencing (Fig. 2C) of the positive clone populations using two main methodologies: Sanger-type sequencing [77] or next-generation sequencing (NGS) [71]. Both approaches offer a direct analysis of clones prior to heterologous scFv expression.
The genes of the clones selected after sequencing can be expressed in different expression systems [72, 78, 79]. In addition, due to its physicochemical characteristics, the scFv is usually produced in large quantities [80]. Validation of the produced molecule can be performed using immunoassays (Fig. 2C) to ensure its complete functionality and use against its target [81, 82]. The use of immunoassays can be applied in the early stages of selection, functioning as a screening strategy for a pre-evaluation of phages fused with the scFv of interest [83, 84]. There are a variety of screening methods that can be used, such as ELISA assays, Western blot and flow cytometry, among others [85].
As an example, a recent study demonstrated that the use of immunoassays was efficient for selecting clones presented by phages against the VP0 protein of human parechovirus 1 (PeVs), an infectious agent associated with several diseases that can affect the gastrointestinal, respiratory and nervous system [86]. In the same study, the sandwich enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunofluorescence were subsequently used to evaluate the binding of the purified scFv molecule to ensure its binding in the native virus. In the study by Zhang
In addition to using ELISA for a preselection of clones, Mansour
scFv Production Methods Using Different Gene Sources
Table 1 presents a detailed compilation of the different types of libraries built and used in the last five years for the production of scFvs using phage display. The libraries are categorized according to their gene origin, size, molecular strategies, target and yield of scFv production.
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Table 1 . Different types of gene libraries built between 2019 and 2024 for the production of scFvs using phage display.
Library Gene source Size Phagomide Fusion Gene Helper Phage Target Yield Reference Immune Chicken 1.7 × 107 pCANTAB5E geneIII M13KO7 N-glycolylneuraminic acid (Neu5Gc) 26.5 mg/l and 36 mg/l [92] Immune Chicken 4 × 106 and 5 × 106 pComb3X geneIII VCS-M13 Coxsackievirus A16 (CA16)__ [93] Immune Chicken 1.5 × 107 pComb3XSS geneIII M13K07 Usutu virus __ [94] Immune Chicken 2.4 × 107 and 6.8 × 107 pComb3X geneIII M13 Protein TS __ [95] Immune Ostrich 2.1 × 108 pSEX81 geneIII M13KO7 Protein tyrosine phosphatase (PTPRN) __ [96] Immune Mouse 1.4 × 1012 pSEX81 geneIII M13KO7 Cathepsin F __ [97] Immune Mouse 1.2 × 107 __ __ M13K07 Human leucine (LRRC15) 1.0-4.5 mg/ml [98] Immune Mouse 1 × 106 and 1 × 108 pHEN1 geneIII __ CD160 and CD123 __ [53] Immune Mouse 1.0 × 107 pSEX81 geneIII M13
KO7ΔpIIINbAAC1-3 __ [99] Immune Mouse 1 × 1011 pIR-DD geneIII M13KO7 Bovine pregnancy-associated glycoprotein (PAG) 3.2 mg/l [100] Immune Rabbit 3 × 106 pComb3X geneIII VSCM13 Ovomucoid protein __ [101] Immune Rabbit 1012 – 1014 pPLFMAΔ25
0pIIIpMMgeneIII VCSM13 IgG human policlonal (huIgG), IgA human (huIgA), CRP and NP-A and NP-B the influenza virus __ [102] Immune Rabbit 3.26 × 109 pIT2 geneIII KM13 Microcystin -LR 3.98 mg/l [103] Immune Horse 10 × 106 and 5 × 107 pCC16 geneIII M13KO7 Native toxins BoNT/A and BoNT/B __ [104] Immune Human 6.12 × 1010 pCDisplay3 geneIII M13K07 Claudine 18.2 __ [105] Immune Human 8.7 × 107 pSEX81 geneIII M13K07 Tetanospasmin 45 μg/ml [106] Immune Human 5 × 1012 pADL geneIII CM13K Receptor-binding domain, (RBD) __ [107] Naïve Human 1 × 1011 pDF geneIII M13K07 Subtilisin/kexin convertase type 9 (PCSK9) 50-200 mg/l [108] Naïve Human 5 × 106 pMod1 geneIII M13K07 Cell HL-60 120 mg/l [109] Synthetic Human 2.5 × 1010 pCANTAB 5E geneIII M13KO7 Immunoglobulin-3 cells T (TIM-3) 10 mg/l [110] Synthetic Human 1.3 × 1010 pComb3X geneIII VCSM13 hNinj1 32 mg/l [111] Semi-synthetic Human 1.47 × 108 pIT2 geneIII M13KO7 Peptide hormone G17-Gly 17.35 mg/l [112] Semi-synthetic Human 1.2 × 1012 pIT2 geneIII M13KO7 Protein PD-1 __ [51] Semi-synthetic Human 2 × 109 pADL-23c __ M13KO7 TNFα, HSA, HEL 50-100 mg/l [113] Semi-synthetic Human 6.8 × 1011 pIT2 geneIII KM13 CCK2R 0.3-1.34 mg/l [114] Note: ( - ) Not informed.
The Production of scFvs Using Immune Libraries
The in vivo immune response can be replicated in vitro through the construction of immune libraries [115]. Immune libraries use the diversity of genes from convalescent patients or immunized donors [116-118]. This strategy has the advantage of in vivo antibody maturation to ensure greater specificity and avidity through the combination of multiple VH and VL variable regions. In the process of maturation, antibody clones undergo somatic hypermutation, selection and clonal expansion, and reach large quantities, which increases the likelihood of the enrichment of high-affinity clones [119]. This causes the gene repertoire to be directed to the antigen used in the immunizations [120].
These libraries use detailed and well-described immunization protocols, which permits their experimental reproducibility. However, although there is great robustness of the method, there are reports of the construction of immune libraries that presented a lower diversity compared to other types of libraries. Rahumatullah
It is worth noting that the immune system is constantly evolving depending on the health status of the host. Thus, immune libraries can offer more than just specific antibodies, but also antibodies derived from memory B cells [122]. This fact implies that there may be rapid responses to antigens already exposed to the body, making the immune response even more effective. Due to the ethical issues related to the immunization of humans and difficulties in obtaining biological samples, different animal models of experimentation, such as chicken [123], rabbit [124], mouse [125], and horse [104], among others, can also be explored regarding their immune response and specificity.
Birds
When compared to mammalian antibodies, avian antibodies, especially those from chickens, have attributes that confer greater experimental versatility in development and applications in immunodiagnostic and immunotherapeutic assays, as well as biophysical, biochemical and bioethical advantages [126]. This is structurally different due to the presence of an additional constant heavy domain, the absence of a region of genuine hinge, and the different composition of oligosaccharides in its lateral chain [127]. At the genetic level, birds possess a single functional V gene to encode the variable region of heavy chains (VH, VH3 family) and light chains (VL, only light chains of type λ) [128]. However, to introduce structural variability, we use alternative mechanisms such as somatic hypermutation and alteration of amino acids in the structural regions (FWRs), responsible for the conformation of the VH and VL chains [129]. The CDR3 region of birds is longer, which can be highly beneficial in the construction of scFvs because longer loops are more stable and have greater sequence diversity and binding capacity to a greater diversity of antigens in comparison to other species [130].
Due to these characteristics, the IgY antibody is constantly being explored to be converted into scFv fragments, becoming a smaller molecule with high specificity [131]. In the work of Wang
In another study, this time by Schoenenwald
Dabiri
Mice
The micés immune system is capable of generating rapid and robust responses against a wide variety of antigens, being considered an effective source for generating antibodies with high specificity and affinity [125]. The general structure and architecture of mouse IgG is similar to human’s, however, small differences in the CDR regions may result in immunogenicity, which may compromise the therapeutic applicability of these antibodies in humans [133]. To reduce immunogenicity, these antibodies can undergo a humanization process. In general terms, humanization comprises different strategies to reduce the immunogenicity of non-human antibodies without losing or drastically altering their functional properties [134]. The main humanization methods are chimerization and grafting of complementarity-determining regions (CDRs).
Chimerization generates antibodies in which the constant regions of murine origin are replaced by human constant regions, and then the residues of the murine variable framework region, except the CDRs, are also replaced by their human equivalents [135]. CDR grafting is the procedure that involves transferring CDRs from a murine “parent” antibody to the framework of a human antibody [136]. Although these humanization methods reduce immunogenicity, it is common to observe a significant drop in antibody affinity for the antigen [134, 137]. Thus, the use of scFvs is a promising alternative since they have desirable therapeutic characteristics and their simple structure allows the exploration of different production and modification strategies to improve their efficacy [138]. Among mammals, mice are often used to construct libraries of scFv. The use of these animals is already well established, with well-described protocols [139]. Most antibody fragments that have been evaluated in preclinical trials originate from these libraries and there are still many molecules that remain under investigation [140].
Recently, Martviset
A study by Baurand
Seeking to overcome the limitations of conventional sequencing methods, Nannini
In the veterinary field, Dormeshkin
Rabbits
In the study by Xu
Rodríguez
Humans
A library named GALBLA1 was built by Effer
To combat tetanus, a disease that primarily plagues children in many developing countries, Nejad
A convalescent COVID-19 patient, who had been infected with SARS-CoV-2 B. 1.617.2 (Delta), provided the gene source for the construction of the immune library in the work of Mendonza-Salazar
The Production of scFvs from Naïve Libraries
In selection strategies in which the objective is to increase the probability of isolation of scFv against, supposedly, any antigen, naïve libraries are also an option that uses the natural repertoire of healthy donors to create a great genetic diversity with good target coverage [141]. These libraries are used to select target-specific ligands independent of donor immune status [115]. Such a feature occurs due to the fact that gene pools of non-immune immunoglobulins are “universal”, in other words, they can contain antibodies to multiple antigens, and therefore it is unnecessary to build a new library for each antigen [5]. This is desirable especially in cases where working with unstable or highly toxic antigens for immunizations occurs [141].
A striking feature of naïve libraries is that the molecules usually have low affinity compared to antibodies isolated from immune libraries; this reinforces the importance of the size of the library generated to ensure greater affinity [142]. The low affinity occurs due to the naïve repertoire not being subjected to the affinity maturation process in vivo, and therefore the production of higher affinity antibodies does not occur [120]. For this reason, when it comes to the relationship of library size with affinity towards the target, it is recommended to building naïve libraries with larger sizes is recommended, precisely to ensure success in isolating antibodies with the highest affinities, since the average size of naïve libraries is in the range of 109 [142].
However, the advantages of the naïve library are the speed of its construction, making it useful in emergency situations, as they do not use experimental animals or long immunization protocols. However, they are able to produce antibodies close to human germline genes with a low risk of immunogenicity [115]. As for its applicability, the naïve library is a preferred type of library for the development of antibodies against infectious agents, such as the bacteria
Healthy Patients
Dong
Erasmus
In the study by Sumphanapai
The Production of scFvs from Synthetic and Semi-Synthetic Libraries
An alternative to the low affinity problem can be solved by synthesizing the scFv artificially. In these cases, synthetic libraries capable of containing billions of functional antibodies are used, whose diversity is derived from the oligonucleotides used in the assembly of the library [149]. Using the sequences available in databases, changes are made in the CDRs of the antibodies obtained and simulations are made to compare them with germline CDR sequences, removing less frequent and undesirable sequences [150]. The revised CDR sequences are assembled to produce the final scFv library [150].
In contrast, the semi-synthetic libraries of scFvs combine only CDRs that were synthesized
The main advantage of the use of
Synthetic scFvs
In the work of Huang
In the work of Bai
Semi-Synthetic scFvs
Valadon
Aghdam
In their study, the target of Ghaderi
Jalilzadeh-Razin
Conclusion
The choice of the gene source is a crucial step for the construction of libraries, as it determines the composition of the scFv gene repertoire and influences characteristics such as diversity, specificity and functionality of the antibody that directly impacts its application. Even after decades of consolidation of the technique for the selection of scFvs using phage display, recent studies have revealed that the strategies used still adopt the use of different libraries for various purposes, which indicates that there is no absolute consensus on the best strategy to be used. However, we noticed a methodological trend regarding the use of immune libraries, despite some related disadvantages, such as their limited diversity. However, the high specificity and affinity of scFvs from these libraries are desirable characteristics that bring efficiency in the selection and relevance for its applicability. We emphasize that there are still challenges to be overcome in the construction of scFvs, which can be overcome by integration with emerging technologies such as genome editing and bioinformatics techniques aimed at increasing quality and specificity. We also highlight that this molecule has enormous potential for high scalability, and is therefore ideal to meet the demands in clinical, medical and industrial research. Finally, we hope that this study will provide a current view of the knowledge about phage gene libraries and the applications of scFvs to guide professionals in the field in the adaptive alignment of their research according to the target studied.
Author Contributions
CCS: conceptualization, investigation, writing - preparation of the original draft; JCG: writing-revision and editing; ERDS: writing-preparation of the original draft; ALGC: visualization; KAGA: writing-revision and editing; IBC: visualization; EVA: writing-revision and editing; LAMM: supervision, writing-revision and editing.
Acknowledgments
The authors would like to thank Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the grants awarded by them.
Conflicts of Interest
The authors have no financial conflicts of interest to declare.
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Published online January 15, 2025 https://doi.org/10.4014/jmb.2407.07049
Copyright © The Korean Society for Microbiology and Biotechnology.
Single-Stranded Variable Fragment Gene Libraries Built for Phage Display: An Updated Review of Design, Selection and Application
Caio Coutinho de Souza1,2, Juliane Corrêa Glória2,3, Eliza Raquel Duarte da Silva2, André de Lima Guerra Corado2,5, Kelson Ávila Graça de Alcântara2,6, Isabelle Bezerra Cordeiro1,7, Edmar Vaz de Andrade1,7, and Luis André Morais Mariúba1,2,3,4,7*
1Programa de Pós-graduação em Biotecnologia (PPGBIOTEC), Universidade Federal do Amazonas (UFAM), Manaus, AM, Brazil
2Laboratório de Diagnóstico e Controle de Doenças Infecciosas na Amazônia (DCDIA), Instituto Leônidas e Maria Deane (ILMD/Fiocruz-Amazônia), Manaus, AM, Brazil
3Programa de Pós-Graduação em Biologia da Interação Patógeno-Hospedeiro (PPGBIO-Interação), Instituto Leônidas e Maria Deane (ILMD/Fiocruz-Amazônia), Manaus, AM, Brazil
4Programa de Pós-graduação em Imunologia Básica e Aplicada (PPGIBA), Universidade Federal do Amazonas (UFAM), Manaus, AM, Brazil
5Universidade Nilton Lins, Manaus, AM, Brazil
6Faculdade Estácio do Amazonas, Manaus, AM, Brazil
7Universidade Federal do Amazonas (UFAM), Manaus, AM, Brazil
Correspondence to:Luis André Morais Mariúba, andre.mariuba@fiocruz.br
Abstract
The development of the phage display technique has brought practicality and speed when selecting high-affinity molecules. It is used to obtain single-chain variable fragments (scFvs) and has revolutionized several branches of research and industry. These are developed from gene libraries that differ in their construction strategies, which causes a diversity of sequences, specificity and binding strength of the projected molecule to its antigen. In this review, we present the recent studies that demonstrate methods and approaches using immune, naïve, synthetic and semi-synthetic libraries to construct and select scFvs. Subsequently, the characteristics of these libraries, the functionality of the scFvs and the cost-benefits of production will be discussed. In addition, we highlight the methodological trends and challenges to be overcome in order to optimize the production and application of these antibody fragments.
Keywords: Gene libraries, phage display, sequence diversity, target affinity, biotechnological applications
Introduction
The researcher George Pieczenik Smith was the first to report in a practical way the use of the phage display technique in 1985, which caused positive impacts in the areas of immunology and molecular biology [1]. It is a method for manipulating the DNA of a bacteriophage to build phage libraries that are capable of encoding molecules to express them in the viral capsid [2].
Currently, this technique is the main molecular tool used to select the single-chain variable fragment (scFv). The scFv is a functional antigen-binding domain containing approximately 30 kDa, formed by the light-chain variable region (VL) and the heavy-chain variable region (VH) and joined by a peptide linker [3]. This antibody format can be rapidly constructed, expressed in different hosts and, due to its reduced size, greater stability and high specificity, has advantages in therapeutic applications and diagnostic tests [4, 5].
Previous studies have reported and evaluated the development and selection of scFvs directed to numerous targets [6–10], which were selected from different gene libraries that differ in origin, construction methods, size and diversity [11]. Constructed libraries can be immune, naïve, synthetic and semi-synthetic in nature [12]. Each approach has its advantages and limitations and, depending on the nature and subsequent use of the ligand analyzed during selection, is suitable for the most varied purposes [13]. Immune libraries require prior immunization of human or animal models with specific antigens, which results in a high affinity for the isolated ligands [14].
Naïve libraries, on the other hand, do not follow classical immunization, since they use a gene repertoire that is usually derived from B cells of donor patients who have not been immunized. However, these are capable of generating molecules with different specificities [15]. Synthetic and semi-synthetic libraries also do not require immunization and can be prepared using bioinformatics analysis to improve the affinity of the molecule, employing random combinatorial mutations in regions responsible for antigen binding [16]. Finally, from any of these possible gene repertoires, the construction of phage libraries, screening and selection of scFv that binds strongly and specifically to its target is performed [17].
Decades after of the emergence of phage display, many advances in this scientific field have already been achieved and are duly documented in a vast extension of studies. However, most reviews on the production of scFvs focus on presenting general aspects of the technique and its applications [18, 19]. Thus, there are no recent reports that describe in a compiled form the diversity, complexity and applicability of the gene libraries used in the process.
The main focus of this review is to provide an up-to-date understanding of the latest research involving scFv gene libraries, in addition to offering guidance to researchers in the field regarding the choice of the best strategy for obtaining scFvs that meet medical and industrial demands. Thus, this review presents the different categories of scFv libraries already developed in the last five years, as well as the origin of the gene repertoire, library size, selection methods, molecular strategies, quality of the selected scFvs, directed targets and the costs versus benefits of the production.
Overcoming Limitations in Recombinant Antibody Generation Using Phage Display
At the end of the nineteenth century, it was found that serum from convalescent human and equine individuals from a bacterial infection could be used to treat this disease, both in humans and animals [20]. However, prolonged use of this therapy caused adverse reactions in patients, probably due to the presence of unknown proteins [21]. In 1960, the structure of antibodies was discovered as scientists sought a better understanding of the mechanisms behind serotherapy [22]. Advances in genetic engineering contributed to improvements in the process of obtaining and purifying antibodies, culminating in the emergence of monoclonal antibodies (mAbs) in 1970 [23]. These mAbs were obtained by hybridoma technology; however, the mAbs produced in murine models when used in human patients were not well tolerated for long periods [24].
The demand for more tolerable options from the clinical and therapeutic point of view fostered several studies with bacteriophages in the 1980 [25]. Bacteriophages are viruses found in nature that have a natural tropism to infect bacterial cells [26]. All the knowledge acquired about bacteriophages helped George Smith to develop the phage display technique when he conducted experiments at the University of Missouri in 1985, research that won him the Nobel Prize in 2018 [26].
In his study, Smith observed that it was possible to insert exogenous DNA next to gene III that encodes a phage surface coat protein, creating a fusion protein expressed in the viral capsid in an accessible and functional way, without compromising the infective activity of the phage [1]. Complementary studies have proven the feasibility of isolating genes of interest from random libraries [27, 28]. Below is a timeline showing the technological advances from the discovery of serotherapy to the realization of the first selections of biotechnological molecules using phage display (Fig. 1).
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Figure 1. Chronology of the main advances from the discovery of the effects of serotherapy to the selection of the first recombinant molecules in phages.
Compared to other methodologies used to obtain therapeutic antibodies, the main advantages of phage display include the specificity of selected clones and the diversity and size of the library that can be generated [29]. The libraries comprise millions of gene sequences, with each phage particle carrying distinct sequences [5].
All the selection is performed in vitro, which promotes the rapid isolation of binding phages in various laboratory environments, thus expanding their usefulness in emergency situations [30]. Many recent studies have described the isolation of scFvs quickly and efficiently against different targets, including in the fight against the SARS-COV-2 virus, the etiological agent responsible for causing the COVID-19 pandemic [31-34].
Although scFv selection occurs in vitro, there are increasing reports of studies that have performed in vivo selections. In these cases, the phage library is injected directly into an animal model, such as mice or rabbits, and circulating phages bind directly to their target in tissues and organs [35, 36]. Phage libraries used in phage display can be stored and preserved for long periods without compromising the capacity for infection, replication, and display of the gene of interest [37]. This high viability allows their reuse and exposure to different targets aiming at the continuous discovery of new molecular interactions [38].
Depending on the chosen molecular strategy, it is possible to join the desired gene to different viral coat proteins (pIII, pVI, pVII pVIII, pIX), which can also have their structure modified to optimize surface display [39-41]. Other elements of the technique are the vectors derived from bacteriophages belonging to the genus
The practicality of the technique has resulted in a considerable number of reviews that have sought to contextualize the general aspects and synthesize knowledge about the improvements that have been achieved [43-46]. The original articles present discoveries of new drugs and therapies [47-49]. Several studies have used bioinformatics strategies to identify and perform specific adjustments in the complementarity determining regions (CDRs) of scFvs involved in the interaction with the antigen [50-53], as well as report combinations of the selection technique with other technologies.
The combination of phage display with hybridoma technology is reported to improve the cloning and expression of the scFv using the genetic information of mammalian germ cell lines [54, 55]. The combination of these two approaches combined the speed and economy of phage display to complement hybridoma technology, thus optimizing time and resources [56].
Dong
Advances in tracking clones of interest have also been achieved. One example is the use of next generation sequencing (NGS) technology, which has been adopted to increase screening and thus identify a larger number of clones from an analyzed library. In the NGS analysis, additional numbers of screened clones are observed when compared to the conventional method, which is performed using colony polymerase chain reaction (PCR) [58]. The integration of phage display with NGS is currently considered a state-of-the-art method, and is indicated for analyzing a substantial amount of gene sequences quickly and effectively [53]. NGS analysis is routinely applied to assess the quality of a library by analyzing changes in heavy chain germline usage and assessing CDR diversity, particularly the composition and length distribution of CDR-H3 [59].
In the work of Krohn
Another important role of NGS in antibody discovery occurs during the methodological process of VH / VL pairing of scFv, which allows the creation of libraries of high diversity and specificity [61]. Choe
In addition, we emphasize that the success of the use of phage display is mainly due to its simplicity, high efficiency, in vitro nature, speed and low cost, which categorizes it as a powerful tool for selecting specific ligands. The evolution of the technique is notorious and new improvements are constantly being made, which makes this technology gain more and more prominence and it contributes significantly to the diagnosis and therapy [63].
The Construction and Expression of scFv
In general, according to the origin of the gene sequence, scFvs can be constructed and selected from immune, naïve, synthetic or semi-synthetic libraries. When the source of the antibody is of an immune or naïve nature, the construction and selection of the scFvs should include the following steps (Fig. 2A): first, mRNA is extracted from the B cells of peripheral blood of human donors, whether healthy or not, or from the blood and lymphoid organs in the case of animals [5]. Then, cDNA synthesis occurs via reverse transcriptase (RT) reactions for amplification of the variable light (VL) and variable heavy (VH) chain segments. It is possible to use specific or random primer pairs that recognize defined or conserved regions, respectively, using PCR [64].
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Figure 2. (A) Library construction steps (B) Screening, selection and elution of binding phages (C) scFv expression and functional evaluation against its target.
The first PCR reaction aims to separately amplify the VH and VL gene. While the second reaction combines the two gene repertoires, which are assembled containing a peptide ligand, usually composed of small and hydrophilic residues of glycine (Gly) and serine (Ser) [65]. PCR is a critical step that directly interferes with cloning, and many studies present strategies for optimizing the steps and reactions to obtain a rapid construction and isolation of scFv [66-68].
During the process of building synthetic and semi-synthetic libraries, neither immunization nor manipulation of biological samples is necessary, since the entire design of the libraries is predicted through bioinformatics analyses. Synthetic libraries are chemically synthesized using oligonucleotides and semi-synthetic libraries are generated by joining synthetic regions with natural regions. These steps are performed to increase the diversity of the CDRs in the assembly process of the VH and VL genes (Fig. 2A).
After assembling the scFv gene from a natural or artificial immune gene source, the next step is to clone it fused to a phage capsid protein gene. For the isolation of high affinity scFvs, fusion to the pIII gene is recommended, as it results in a more controlled and precise presentation [4]. Among the available vector options, the pcomb3 family of vectors are preferable for the generation and selection of antibody fragment libraries.
A competent bacterium such as
Candidates identified in the first round of selection may present low affinity due to some biases such as the propagation of transforming bacteria that received the vector with the scFv gene of interest, the reduced efficiency of infection and replication of phages in the host bacteria, in addition to competition of positive clones with large numbers of mutant clones with weak affinity to the target [73, 74]. To identify high-affinity scFvs, it is recommended to perform a gradual increase in the number of screening rounds, usually, two to four rounds are necessary. This process progressively removes non-binding phages and enriches the population of phages with a higher binding strength [56].
Other parameters that affect the efficiency of biopanning screening include the concentration of immobilized antigen, the concentration of the non-ionic detergent used in the washes, and the composition of the blocking buffer [75]. Such factors require careful methodological balance, as low-stringency screening may allow retention of false positives, while high-stringency screening may result in the removal of positive clones.
The success of the bioprospection depends on the quality and format of the antigen presentation, which is crucial in the isolation of scFvs, and requires the establishment of different biopanning strategies [38, 76]. A recent study by Kamstrup
At the end of the selection rounds, although the selected molecules are not monoclonal in origin, they will have high affinity and binding strength comparable to or superior to conventional mAbs [56]. The final steps consist of sequencing (Fig. 2C) of the positive clone populations using two main methodologies: Sanger-type sequencing [77] or next-generation sequencing (NGS) [71]. Both approaches offer a direct analysis of clones prior to heterologous scFv expression.
The genes of the clones selected after sequencing can be expressed in different expression systems [72, 78, 79]. In addition, due to its physicochemical characteristics, the scFv is usually produced in large quantities [80]. Validation of the produced molecule can be performed using immunoassays (Fig. 2C) to ensure its complete functionality and use against its target [81, 82]. The use of immunoassays can be applied in the early stages of selection, functioning as a screening strategy for a pre-evaluation of phages fused with the scFv of interest [83, 84]. There are a variety of screening methods that can be used, such as ELISA assays, Western blot and flow cytometry, among others [85].
As an example, a recent study demonstrated that the use of immunoassays was efficient for selecting clones presented by phages against the VP0 protein of human parechovirus 1 (PeVs), an infectious agent associated with several diseases that can affect the gastrointestinal, respiratory and nervous system [86]. In the same study, the sandwich enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunofluorescence were subsequently used to evaluate the binding of the purified scFv molecule to ensure its binding in the native virus. In the study by Zhang
In addition to using ELISA for a preselection of clones, Mansour
scFv Production Methods Using Different Gene Sources
Table 1 presents a detailed compilation of the different types of libraries built and used in the last five years for the production of scFvs using phage display. The libraries are categorized according to their gene origin, size, molecular strategies, target and yield of scFv production.
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Table 1 . Different types of gene libraries built between 2019 and 2024 for the production of scFvs using phage display..
Library Gene source Size Phagomide Fusion Gene Helper Phage Target Yield Reference Immune Chicken 1.7 × 107 pCANTAB5E geneIII M13KO7 N-glycolylneuraminic acid (Neu5Gc) 26.5 mg/l and 36 mg/l [92] Immune Chicken 4 × 106 and 5 × 106 pComb3X geneIII VCS-M13 Coxsackievirus A16 (CA16)__ [93] Immune Chicken 1.5 × 107 pComb3XSS geneIII M13K07 Usutu virus __ [94] Immune Chicken 2.4 × 107 and 6.8 × 107 pComb3X geneIII M13 Protein TS __ [95] Immune Ostrich 2.1 × 108 pSEX81 geneIII M13KO7 Protein tyrosine phosphatase (PTPRN) __ [96] Immune Mouse 1.4 × 1012 pSEX81 geneIII M13KO7 Cathepsin F __ [97] Immune Mouse 1.2 × 107 __ __ M13K07 Human leucine (LRRC15) 1.0-4.5 mg/ml [98] Immune Mouse 1 × 106 and 1 × 108 pHEN1 geneIII __ CD160 and CD123 __ [53] Immune Mouse 1.0 × 107 pSEX81 geneIII M13
KO7ΔpIIINbAAC1-3 __ [99] Immune Mouse 1 × 1011 pIR-DD geneIII M13KO7 Bovine pregnancy-associated glycoprotein (PAG) 3.2 mg/l [100] Immune Rabbit 3 × 106 pComb3X geneIII VSCM13 Ovomucoid protein __ [101] Immune Rabbit 1012 – 1014 pPLFMAΔ25
0pIIIpMMgeneIII VCSM13 IgG human policlonal (huIgG), IgA human (huIgA), CRP and NP-A and NP-B the influenza virus __ [102] Immune Rabbit 3.26 × 109 pIT2 geneIII KM13 Microcystin -LR 3.98 mg/l [103] Immune Horse 10 × 106 and 5 × 107 pCC16 geneIII M13KO7 Native toxins BoNT/A and BoNT/B __ [104] Immune Human 6.12 × 1010 pCDisplay3 geneIII M13K07 Claudine 18.2 __ [105] Immune Human 8.7 × 107 pSEX81 geneIII M13K07 Tetanospasmin 45 μg/ml [106] Immune Human 5 × 1012 pADL geneIII CM13K Receptor-binding domain, (RBD) __ [107] Naïve Human 1 × 1011 pDF geneIII M13K07 Subtilisin/kexin convertase type 9 (PCSK9) 50-200 mg/l [108] Naïve Human 5 × 106 pMod1 geneIII M13K07 Cell HL-60 120 mg/l [109] Synthetic Human 2.5 × 1010 pCANTAB 5E geneIII M13KO7 Immunoglobulin-3 cells T (TIM-3) 10 mg/l [110] Synthetic Human 1.3 × 1010 pComb3X geneIII VCSM13 hNinj1 32 mg/l [111] Semi-synthetic Human 1.47 × 108 pIT2 geneIII M13KO7 Peptide hormone G17-Gly 17.35 mg/l [112] Semi-synthetic Human 1.2 × 1012 pIT2 geneIII M13KO7 Protein PD-1 __ [51] Semi-synthetic Human 2 × 109 pADL-23c __ M13KO7 TNFα, HSA, HEL 50-100 mg/l [113] Semi-synthetic Human 6.8 × 1011 pIT2 geneIII KM13 CCK2R 0.3-1.34 mg/l [114] Note: ( - ) Not informed..
The Production of scFvs Using Immune Libraries
The in vivo immune response can be replicated in vitro through the construction of immune libraries [115]. Immune libraries use the diversity of genes from convalescent patients or immunized donors [116-118]. This strategy has the advantage of in vivo antibody maturation to ensure greater specificity and avidity through the combination of multiple VH and VL variable regions. In the process of maturation, antibody clones undergo somatic hypermutation, selection and clonal expansion, and reach large quantities, which increases the likelihood of the enrichment of high-affinity clones [119]. This causes the gene repertoire to be directed to the antigen used in the immunizations [120].
These libraries use detailed and well-described immunization protocols, which permits their experimental reproducibility. However, although there is great robustness of the method, there are reports of the construction of immune libraries that presented a lower diversity compared to other types of libraries. Rahumatullah
It is worth noting that the immune system is constantly evolving depending on the health status of the host. Thus, immune libraries can offer more than just specific antibodies, but also antibodies derived from memory B cells [122]. This fact implies that there may be rapid responses to antigens already exposed to the body, making the immune response even more effective. Due to the ethical issues related to the immunization of humans and difficulties in obtaining biological samples, different animal models of experimentation, such as chicken [123], rabbit [124], mouse [125], and horse [104], among others, can also be explored regarding their immune response and specificity.
Birds
When compared to mammalian antibodies, avian antibodies, especially those from chickens, have attributes that confer greater experimental versatility in development and applications in immunodiagnostic and immunotherapeutic assays, as well as biophysical, biochemical and bioethical advantages [126]. This is structurally different due to the presence of an additional constant heavy domain, the absence of a region of genuine hinge, and the different composition of oligosaccharides in its lateral chain [127]. At the genetic level, birds possess a single functional V gene to encode the variable region of heavy chains (VH, VH3 family) and light chains (VL, only light chains of type λ) [128]. However, to introduce structural variability, we use alternative mechanisms such as somatic hypermutation and alteration of amino acids in the structural regions (FWRs), responsible for the conformation of the VH and VL chains [129]. The CDR3 region of birds is longer, which can be highly beneficial in the construction of scFvs because longer loops are more stable and have greater sequence diversity and binding capacity to a greater diversity of antigens in comparison to other species [130].
Due to these characteristics, the IgY antibody is constantly being explored to be converted into scFv fragments, becoming a smaller molecule with high specificity [131]. In the work of Wang
In another study, this time by Schoenenwald
Dabiri
Mice
The micés immune system is capable of generating rapid and robust responses against a wide variety of antigens, being considered an effective source for generating antibodies with high specificity and affinity [125]. The general structure and architecture of mouse IgG is similar to human’s, however, small differences in the CDR regions may result in immunogenicity, which may compromise the therapeutic applicability of these antibodies in humans [133]. To reduce immunogenicity, these antibodies can undergo a humanization process. In general terms, humanization comprises different strategies to reduce the immunogenicity of non-human antibodies without losing or drastically altering their functional properties [134]. The main humanization methods are chimerization and grafting of complementarity-determining regions (CDRs).
Chimerization generates antibodies in which the constant regions of murine origin are replaced by human constant regions, and then the residues of the murine variable framework region, except the CDRs, are also replaced by their human equivalents [135]. CDR grafting is the procedure that involves transferring CDRs from a murine “parent” antibody to the framework of a human antibody [136]. Although these humanization methods reduce immunogenicity, it is common to observe a significant drop in antibody affinity for the antigen [134, 137]. Thus, the use of scFvs is a promising alternative since they have desirable therapeutic characteristics and their simple structure allows the exploration of different production and modification strategies to improve their efficacy [138]. Among mammals, mice are often used to construct libraries of scFv. The use of these animals is already well established, with well-described protocols [139]. Most antibody fragments that have been evaluated in preclinical trials originate from these libraries and there are still many molecules that remain under investigation [140].
Recently, Martviset
A study by Baurand
Seeking to overcome the limitations of conventional sequencing methods, Nannini
In the veterinary field, Dormeshkin
Rabbits
In the study by Xu
Rodríguez
Humans
A library named GALBLA1 was built by Effer
To combat tetanus, a disease that primarily plagues children in many developing countries, Nejad
A convalescent COVID-19 patient, who had been infected with SARS-CoV-2 B. 1.617.2 (Delta), provided the gene source for the construction of the immune library in the work of Mendonza-Salazar
The Production of scFvs from Naïve Libraries
In selection strategies in which the objective is to increase the probability of isolation of scFv against, supposedly, any antigen, naïve libraries are also an option that uses the natural repertoire of healthy donors to create a great genetic diversity with good target coverage [141]. These libraries are used to select target-specific ligands independent of donor immune status [115]. Such a feature occurs due to the fact that gene pools of non-immune immunoglobulins are “universal”, in other words, they can contain antibodies to multiple antigens, and therefore it is unnecessary to build a new library for each antigen [5]. This is desirable especially in cases where working with unstable or highly toxic antigens for immunizations occurs [141].
A striking feature of naïve libraries is that the molecules usually have low affinity compared to antibodies isolated from immune libraries; this reinforces the importance of the size of the library generated to ensure greater affinity [142]. The low affinity occurs due to the naïve repertoire not being subjected to the affinity maturation process in vivo, and therefore the production of higher affinity antibodies does not occur [120]. For this reason, when it comes to the relationship of library size with affinity towards the target, it is recommended to building naïve libraries with larger sizes is recommended, precisely to ensure success in isolating antibodies with the highest affinities, since the average size of naïve libraries is in the range of 109 [142].
However, the advantages of the naïve library are the speed of its construction, making it useful in emergency situations, as they do not use experimental animals or long immunization protocols. However, they are able to produce antibodies close to human germline genes with a low risk of immunogenicity [115]. As for its applicability, the naïve library is a preferred type of library for the development of antibodies against infectious agents, such as the bacteria
Healthy Patients
Dong
Erasmus
In the study by Sumphanapai
The Production of scFvs from Synthetic and Semi-Synthetic Libraries
An alternative to the low affinity problem can be solved by synthesizing the scFv artificially. In these cases, synthetic libraries capable of containing billions of functional antibodies are used, whose diversity is derived from the oligonucleotides used in the assembly of the library [149]. Using the sequences available in databases, changes are made in the CDRs of the antibodies obtained and simulations are made to compare them with germline CDR sequences, removing less frequent and undesirable sequences [150]. The revised CDR sequences are assembled to produce the final scFv library [150].
In contrast, the semi-synthetic libraries of scFvs combine only CDRs that were synthesized
The main advantage of the use of
Synthetic scFvs
In the work of Huang
In the work of Bai
Semi-Synthetic scFvs
Valadon
Aghdam
In their study, the target of Ghaderi
Jalilzadeh-Razin
Conclusion
The choice of the gene source is a crucial step for the construction of libraries, as it determines the composition of the scFv gene repertoire and influences characteristics such as diversity, specificity and functionality of the antibody that directly impacts its application. Even after decades of consolidation of the technique for the selection of scFvs using phage display, recent studies have revealed that the strategies used still adopt the use of different libraries for various purposes, which indicates that there is no absolute consensus on the best strategy to be used. However, we noticed a methodological trend regarding the use of immune libraries, despite some related disadvantages, such as their limited diversity. However, the high specificity and affinity of scFvs from these libraries are desirable characteristics that bring efficiency in the selection and relevance for its applicability. We emphasize that there are still challenges to be overcome in the construction of scFvs, which can be overcome by integration with emerging technologies such as genome editing and bioinformatics techniques aimed at increasing quality and specificity. We also highlight that this molecule has enormous potential for high scalability, and is therefore ideal to meet the demands in clinical, medical and industrial research. Finally, we hope that this study will provide a current view of the knowledge about phage gene libraries and the applications of scFvs to guide professionals in the field in the adaptive alignment of their research according to the target studied.
Author Contributions
CCS: conceptualization, investigation, writing - preparation of the original draft; JCG: writing-revision and editing; ERDS: writing-preparation of the original draft; ALGC: visualization; KAGA: writing-revision and editing; IBC: visualization; EVA: writing-revision and editing; LAMM: supervision, writing-revision and editing.
Acknowledgments
The authors would like to thank Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the grants awarded by them.
Conflicts of Interest
The authors have no financial conflicts of interest to declare.
- Abstract
- Introduction
- Overcoming Limitations in Recombinant Antibody Generation Using Phage Display
- The Construction and Expression of scFv
- scFv Production Methods Using Different Gene Sources
- The Production of scFvs Using Immune Libraries
- Birds
- Mice
- Rabbits
- Humans
- The Production of scFvs from Naïve Libraries
- Healthy Patients
- The Production of scFvs from Synthetic and Semi-Synthetic Libraries
- Synthetic scFvs
- Semi-Synthetic scFvs
- Conclusion
- Author Contributions
- Acknowledgments
- Conflicts of Interest
Fig 1.

Fig 2.

-
Table 1 . Different types of gene libraries built between 2019 and 2024 for the production of scFvs using phage display..
Library Gene source Size Phagomide Fusion Gene Helper Phage Target Yield Reference Immune Chicken 1.7 × 107 pCANTAB5E geneIII M13KO7 N-glycolylneuraminic acid (Neu5Gc) 26.5 mg/l and 36 mg/l [92] Immune Chicken 4 × 106 and 5 × 106 pComb3X geneIII VCS-M13 Coxsackievirus A16 (CA16)__ [93] Immune Chicken 1.5 × 107 pComb3XSS geneIII M13K07 Usutu virus __ [94] Immune Chicken 2.4 × 107 and 6.8 × 107 pComb3X geneIII M13 Protein TS __ [95] Immune Ostrich 2.1 × 108 pSEX81 geneIII M13KO7 Protein tyrosine phosphatase (PTPRN) __ [96] Immune Mouse 1.4 × 1012 pSEX81 geneIII M13KO7 Cathepsin F __ [97] Immune Mouse 1.2 × 107 __ __ M13K07 Human leucine (LRRC15) 1.0-4.5 mg/ml [98] Immune Mouse 1 × 106 and 1 × 108 pHEN1 geneIII __ CD160 and CD123 __ [53] Immune Mouse 1.0 × 107 pSEX81 geneIII M13
KO7ΔpIIINbAAC1-3 __ [99] Immune Mouse 1 × 1011 pIR-DD geneIII M13KO7 Bovine pregnancy-associated glycoprotein (PAG) 3.2 mg/l [100] Immune Rabbit 3 × 106 pComb3X geneIII VSCM13 Ovomucoid protein __ [101] Immune Rabbit 1012 – 1014 pPLFMAΔ25
0pIIIpMMgeneIII VCSM13 IgG human policlonal (huIgG), IgA human (huIgA), CRP and NP-A and NP-B the influenza virus __ [102] Immune Rabbit 3.26 × 109 pIT2 geneIII KM13 Microcystin -LR 3.98 mg/l [103] Immune Horse 10 × 106 and 5 × 107 pCC16 geneIII M13KO7 Native toxins BoNT/A and BoNT/B __ [104] Immune Human 6.12 × 1010 pCDisplay3 geneIII M13K07 Claudine 18.2 __ [105] Immune Human 8.7 × 107 pSEX81 geneIII M13K07 Tetanospasmin 45 μg/ml [106] Immune Human 5 × 1012 pADL geneIII CM13K Receptor-binding domain, (RBD) __ [107] Naïve Human 1 × 1011 pDF geneIII M13K07 Subtilisin/kexin convertase type 9 (PCSK9) 50-200 mg/l [108] Naïve Human 5 × 106 pMod1 geneIII M13K07 Cell HL-60 120 mg/l [109] Synthetic Human 2.5 × 1010 pCANTAB 5E geneIII M13KO7 Immunoglobulin-3 cells T (TIM-3) 10 mg/l [110] Synthetic Human 1.3 × 1010 pComb3X geneIII VCSM13 hNinj1 32 mg/l [111] Semi-synthetic Human 1.47 × 108 pIT2 geneIII M13KO7 Peptide hormone G17-Gly 17.35 mg/l [112] Semi-synthetic Human 1.2 × 1012 pIT2 geneIII M13KO7 Protein PD-1 __ [51] Semi-synthetic Human 2 × 109 pADL-23c __ M13KO7 TNFα, HSA, HEL 50-100 mg/l [113] Semi-synthetic Human 6.8 × 1011 pIT2 geneIII KM13 CCK2R 0.3-1.34 mg/l [114] Note: ( - ) Not informed..
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