Microfluidics, an effective tool for supporting phage display-A review.

Phage display is a vital tool for the discovery and development of affinity reagents such as antibodies and peptides, which have great potential in imaging, molecular recognition, biosensors, targeted delivery and other clinical applications. However, affinity reagents obtained by phage display are often subjected to a process called biopanning, which is considered time-consuming, labor-intensive and lacks accurate control, limiting the acquisition of high-quality affinity reagents. Over the last two decades, several microfluidic approaches have been designed to simplify the conventional biopanning process and to realize precise control. To better understand the advantages of microfluidics over traditional biopanning and the potential of microfluidics for other molecular screening strategies, we provided an overview of recent applications of microfluidics in phage display. Additionally, the next challenges and outlooks are discussed.

Biosensing strategies using recombinant luminescent proteins and their use for food and environmental analysis.

Progress in synthetic biology and nanotechnology plays at present a major role in the fabrication of sophisticated and miniaturized analytical devices that provide the means to tackle the need for new tools and methods for environmental and food safety. Significant research efforts have led to biosensing experiments experiencing a remarkable growth with the development and application of recombinant luminescent proteins (RLPs) being at the core of this boost. Integrating RLPs into biosensors has resulted in highly versatile detection platforms. These platforms include luminescent enzyme-linked immunosorbent assays (ELISAs), bioluminescence resonance energy transfer (BRET)-based sensors, and genetically encoded luminescent biosensors. Increased signal-to-noise ratios, rapid response times, and the ability to monitor dynamic biological processes in live cells are advantages inherent to the approaches mentioned above. Furthermore, novel fusion proteins and optimized expression systems to improve their stability, brightness, and spectral properties have enhanced the performance and pertinence of luminescent biosensors in diverse fields. This review highlights recent progress in RLP-based biosensing, showcasing their implementation for monitoring different contaminants commonly found in food and environmental samples. Future perspectives and potential challenges in these two areas of interest are also addressed, providing a comprehensive overview of the current state and a forecast of the biosensing strategies using recombinant luminescent proteins to come.

Neutralizing chimeric heavy-chain antibody targeting the L-HN domain of Clostridium botulinum neurotoxin type F.

Botulinum toxin (BoNT) from Clostridium botulinum is the most toxic biotoxin known and is also an important bioterrorism agent. After poisoning, the only effective treatment is injection of antitoxin. However, neutralizing nanoantibodies are safer and more effective, representing a promising therapeutic approach. Therefore, it is important to obtain effective neutralizing nanoantibodies. Hence, the present study aimed to construct a phage antibody library by immunizing a camel and screening specific clones that bind to the L-HN domain of BoNT/F and constructing chimeric heavy-chain antibodies by fusing them with a human Fc fragment. The antibodies' affinity and in vivo neutralizing activities were evaluated. The results showed that 2 µg of F20 antibody could completely neutralize 20 × the median lethal dose (LD50) of BoNT/F in vitro. Injection of 5 mg/kg F20 at 1 h, 2 h, 3 h, and 4 h into mice after BoNT/F challenge resulted in complete survival in vivo. Overall, the antibody might be a candidate for the development of new drugs to treat botulism.

Screening and identification of nanobody against inhibin α-subunit from a Camelus bactrianus phage display library.

Background: Inhibin is a member of the transforming growth factor family that influences reproduction in animals. Objective: The purpose of this study was to obtain nanobodies from the phage antibody library constructed by us that can specifically bind to inhibin α-subunit. Methods: In this study, camels were immunized with Kazakh sheep inhibin-α protein that expressed in BL21 E. coli, and the camel VHH nanobody phage display library was prepared using nested PCR. The nanobodies specifically binding to inhibin α-subunit in the library were screened by three rounds of immunoaffinity screening and phage enzyme-linked immunosorbent assay (phage ELISA). The functions of the selected nanobodies were identified using molecular simulation docking, ELISA affinity test, and sheep immunity test. Results: A nanobody display library was successfully constructed with a capacity of 1.05 × 1012 CFU, and four inhibin-α-subunit-specific nanobodies with an overall similarity of 69.34 % were screened from the library, namely, Nb-4, Nb-15, Nb-26, and Nb-57. The results of molecular simulation docking revealed that four types of nanobodies were complexed with inhibin-α protein mainly through hydrophobic bonds. Immunity tests revealed that the nanobody Nb-4 could effectively inhibit sheep inhibin A/B and could significantly improve the FSH level in sheep. Conclusion: Four inhibin α-subunit-specific nanobodies with biological functions were successfully screened. To the best of our knowledge, this is a new reproductive immunomodulatory pathway of inhibin α-subunit, which may change the secretion of FSH in the ovary, thus changing the estrous cycle of organisms.

Molecular dynamics simulation of the brain-isolated single-domain antibody/nanobody from camels through in vivo phage display screening.

Introduction: During the last decade, there has been a significant rise in the use of therapeutic antibodies or passive immunotherapy for treating various conditions like inflammation and cancer. However, these proteins face challenges reaching the brain and often require specialized delivery methods such as single-domain antibodies (sdAbs). Traditional antibodies struggle to efficiently cross the blood-brain barrier (BBB), hindering their effectiveness. Receptor-mediated transcytosis (RMT) offers a promising pathway for transporting large molecules essential for brain function and treatment across the BBB. Methods: SdAbs and peptide ligands with an affinity for RMT receptors are commonly employed to enhance the transport of biotherapeutics compounds across the BBB. This research used a sdAbs phage-displayed library from 13 camelus dromedarius samples to identify sdABs that specifically bind to and are internalized by human BBB endothelial cells (ECs) through in vivo panning. Results and discussion: One sdAb, defined as FB24, was isolated, sequenced, translated into an open reading frame (ORF), and subjected to three-dimensional (3D) modeling. Molecular docking and molecular dynamics simulations were carried out by the HADDOCK web server and GROMACS, respectively, to evaluate the interaction between FB24 and EC receptors in silico. The docking results revealed that FB24 exhibited binding activity against potential EC receptors with -1.7 to -2.7 ranged z score and maintained a stable structure. The docked complex of FB24-RAGE (receptor for advanced glycation end products, also known as advanced glycation end product receptor [AGER]) showed 18 hydrogen bonds and 213 non-bonded contacts. It was chosen for further analysis by molecular dynamics simulations by GROMACS. This complex showed a stable condition, and its root mean square deviation (RMSD) was 0.218 nm. The results suggest that FB24 could serve as a suitable carrier vector for transporting therapeutic and diagnostic agents across the BBB to the brain through a non-invasive route.

Encoding and display technologies for combinatorial libraries in drug discovery: The coming of age from biology to therapy.

Drug discovery is a sophisticated process that incorporates scientific innovations and cutting-edge technologies. Compared to traditional bioactivity-based screening methods, encoding and display technologies for combinatorial libraries have recently advanced from proof-of-principle experiments to promising tools for pharmaceutical hit discovery due to their high screening efficiency, throughput, and resource minimization. This review systematically summarizes the development history, typology, and prospective applications of encoding and displayed technologies, including phage display, ribosomal display, mRNA display, yeast cell display, one-bead one-compound, DNA-encoded, peptide nucleic acid-encoded, and new peptide-encoded technologies, and examples of preclinical and clinical translation. We discuss the progress of novel targeted therapeutic agents, covering a spectrum from small-molecule inhibitors and nonpeptidic macrocycles to linear, monocyclic, and bicyclic peptides, in addition to antibodies. We also address the pending challenges and future prospects of drug discovery, including the size of screening libraries, advantages and disadvantages of the technology, clinical translational potential, and market space. This review is intended to establish a comprehensive high-throughput drug discovery strategy for scientific researchers and clinical drug developers.

Screening and affinity optimization of single domain antibody targeting the SARS-CoV-2 nucleocapsid protein.

The coronavirus disease 2019 (COVID-19) pandemic, which caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lead to a crisis with devastating disasters to global public economy and health. Several studies suggest that the SARS-CoV-2 nucleocapsid protein (N protein) is one of uppermost structural constituents of SARS-CoV-2 and is relatively conserved which could become a specific diagnostic marker. In this study, eight single domain antibodies recognized the N protein specifically which were named pN01-pN08 were screened using human phage display library. According to multiple sequence alignment and molecular docking analyses, the interaction mechanism between antibody and N protein was predicted. ELISA results indicated pN01-pN08 with high affinity to protein N. To improve their efficacy, two fusion proteins were prepared and their affinity was tested. These finding showed that fusion proteins had higher affinity than single domain antibodies and will be used as diagnosis for the pandemic of SARS-CoV-2.

Phage Immunoprecipitation and Sequencing-a Versatile Technique for Mapping the Antibody Reactome.

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies, and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.

Selective Clearance of Circulating Histones Based on Dodecapeptide-Grafted Copolymer Material for Sepsis Blood Purification.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Research indicates that circulating histones, as pathogenic factors, may represent a therapeutic target for sepsis. However, effectively clearing circulating histones poses a challenge due to their structural similarity to normal blood proteins, their low abundance in the bloodstream, and serious interference from other blood biomacromolecules. Here we design a dodecapeptide-based functional polymer that can selectively adsorb circulating histones from the blood. The peptide, named P1 (HNHHQLALVESY), was discovered through phage display screening and demonstrated a strong affinity for circulating histones while exhibiting negligible affinities for common proteins in the blood, such as human serum albumin (HSA), immunoglobulin G (IgG), and transferrin (TRF). Furthermore, the P1 peptide was incorporated into a functional polymer design, poly(PEGMA-co-P1), which was immobilized onto a silica gel surface through reversible addition-fragmentation chain transfer polymerization. The resulting material was characterized using solid nuclear magnetic resonance, thermogravimetric analysis, and X-ray photoelectron spectroscopy. This material demonstrated the ability to selectively and efficiently capture circulating histones from both model solutions and whole blood samples while also exhibiting satisfactory blood compatibility, good antifouling properties, and resistance to interference. Satisfactory binding affinity and efficient capture capacity toward histone were also observed for the other screened peptide P2 (QMSMDLFGSNFV)-grafted polymer, validating phage display as a reliable ligand screening strategy. These findings present an approach for the specific clearance of circulating histones and hold promise for future clinical applications in blood purification toward sepsis.

Identification of Conserved Linear Epitopes on Viral Protein 2 of Foot-and-Mouth Disease Virus Serotype O by Monoclonal Antibodies 6F4.D11.B6 and 8D6.B9.C3.

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide essential information for developing diagnostic assays and vaccine designs. In this study, monoclonal antibodies raised against FMD virus (FMDV) were produced. Sixty-six monoclonal antibodies demonstrated strong reactivity and specificity to FMDV. The purified monoclonal antibodies were further used for bio-panning to select phage expressing specific epitopes from phage-displayed 12 mer-peptide library. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. Two hybridoma clones secreted monoclonal antibodies recognizing linear epitopes on VP2 of FMDV serotype O. The non-neutralizing monoclonal antibody 6F4.D11.B6 recognized the residues 67-78 on antigenic site 2 resinding in VP2, while the neutralizing monoclonal antibody 8D6.B9.C3 recognized a novel linear epitope encompassing residues 115-126 on VP2. This information and the FMDV-specific monoclonal antibodies provide valuable sources for further study and application in diagnosis, therapeutics and vaccine designs to strengthen the disease prevention and control measures.

A next-generation Fab library platform directly yielding drug-like antibodies with high affinity, diversity, and developability.

We previously described an in vitro single-chain fragment (scFv) library platform originally designed to generate antibodies with excellent developability properties. The platform design was based on the use of clinical antibodies as scaffolds into which replicated natural complementarity-determining regions purged of sequence liabilities were inserted, and the use of phage and yeast display to carry out antibody selection. In addition to being developable, antibodies generated using our platform were extremely diverse, with most campaigns yielding sub-nanomolar binders. Here, we describe a platform advancement that incorporates Fab phage display followed by single-chain antibody-binding fragment Fab (scFab) yeast display. The scFab single-gene format provides balanced expression of light and heavy chains, with enhanced conversion to IgG, thereby combining the advantages of scFvs and Fabs. A meticulously engineered, quality-controlled Fab phage library was created using design principles similar to those used to create the scFv library. A diverse panel of binding scFabs, with high conversion efficiency to IgG, was isolated against two targets. This study highlights the compatibility of phage and yeast display with a Fab semi-synthetic library design, offering an efficient approach to generate drug-like antibodies directly, facilitating their conversion to potential therapeutic candidates.

68Ga Radiolabeling of NODASA-Functionalized Phage Display-Derived Peptides for Prospective Assessment as Tuberculosis-Specific PET Radiotracers.

This research presents the development of positron emission tomography (PET) radiotracers for detecting Mycobacterium tuberculosis (MTB) for the diagnosis and monitoring of tuberculosis. Two phage display-derived peptides with proven selective binding to MTB were identified for development into PET radiopharmaceuticals: H8 (linear peptide) and PH1 (cyclic peptide). We sought to functionalize H8/PH1 with NODASA, a bifunctional chelator that allows complexation of PET-compatible radiometals such as gallium-68. Herein, we report on the chelator functionalization, optimized radiosynthesis, and assessment of the radiopharmaceutical properties of [68Ga]Ga-NODASA-H8 and [68Ga]Ga-NODASA-PH1. Robust radiolabeling was achieved using the established routine method, indicating consistent production of a radiochemically pure product (RCP ≥ 99.6%). For respective [68Ga]Ga-NODASA-H8 and [68Ga]Ga-NODASA-PH1, relatively high levels of decay-corrected radiochemical yield (91.2% ± 2.3%, 86.7% ± 4.0%) and apparent molar activity (Am, 3.9 ± 0.8 and 34.0 ± 5.3 GBq/µmol) were reliably achieved within 42 min, suitable for imaging purposes. Notably, [68Ga]Ga-NODASA-PH1 remained stable in blood plasma for up to 2 h, while [68Ga]Ga-NODASA-H8 degraded within 30 min. For both 68Ga peptides, minimal whole-blood cell binding and plasma protein binding were observed, indicating a favorable pharmaceutical behavior. [68Ga]Ga-NODASA-PH1 is a promising candidate for further in vitro/in vivo evaluation as a tuberculosis-specific infection imaging agent.

Rapid and sensitive detection of SARS-CoV-2 based on a phage-displayed scFv antibody fusion with alkaline phosphatase and NanoLuc luciferase.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses.

Synthetic antibodies for accelerated RNA crystallography.

RNA structure is crucial to a wide range of cellular processes. The intimate relationship between macromolecular structure and function necessitates the determination of high-resolution structures of functional RNA molecules. X-ray crystallography is the predominant technique used for macromolecular structure determination; however, solving RNA structures has been more challenging than their protein counterparts, as reflected in their poor representation in the Protein Data Bank (<1%). Antibody-assisted RNA crystallography is a relatively new technique that promises to accelerate RNA structure determination by employing synthetic antibodies (Fabs) as crystallization chaperones that are specifically raised against target RNAs. Antibody chaperones facilitate the formation of ordered crystal lattices by minimizing RNA flexibility and replacing unfavorable RNA-RNA contacts with contacts between chaperone molecules. Atomic coordinates of these antibody fragments can also be used as search models to obtain phase information during structure determination. Antibody-assisted RNA crystallography has enabled the structure determination of 15 unique RNA targets, including 11 in the last 6 years. In this review, I cover the historical development of antibody fragments as crystallization chaperones and their application to diverse RNA targets. I discuss how the first structures of antibody-RNA complexes informed the design of second-generation antibodies and led to the development of portable crystallization modules that have greatly reduced the uncertainties associated with RNA crystallography. Finally, I outline unexplored avenues that can increase the impact of this technology in structural biology research and discuss potential applications of antibodies as affinity reagents for interrogating RNA biology outside of their use in crystallography. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Evaluation of human antibodies from vaccinated volunteers for protection against Yersinia pestis infection.

Yersinia pestis has a broad host range and has caused lethal bubonic and pneumonic plague in humans. With the emergence of multiple resistant strains and the potential for biothreat use, there is an urgent need for new therapeutic strategies that can protect populations from natural or deliberate infection. Targeting F1 has been proven to be the main strategy for developing vaccines and therapeutic antibodies, but data on anti-F1 antibodies, especially in humans, are scarce. To date, three human anti-F1 monoclonal antibodies (m252, αF1Ig2, and αF1Ig8) from naive populations have been reported. Here, we constructed an antibody library from vaccinees immunized with the plague subunit vaccine IIa by phage display. The genetic basis, epitopes, and biological functions of the obtained mAbs were assessed and evaluated in plague-challenged mice. Three human mAbs, namely, F3, F19, and F23, were identified. Their biolayer responses were 0.4, 0.6, and 0.6 nm, respectively. The dissociation constants (KD) of the F1 antigen were 1 pM, 0.165 nM, and 1 pM, respectively. Although derived from distinct Ab lineages, that is, VH3-30-D3-10-JH4 (F3&F23) and VH3-43-D6-19-JH4 (F19), these mAbs share similar binding sites in F1 with some overlap with αF1Ig8 but are distinct from αF1Ig2. Each of them provided a significant protective effect for Balb/c mice against a 100 median lethal dose (MLD) challenge of a virulent Y. pestis strain when administered at a dose of 100 µg. No synergistic or antagonistic effects were observed among them. These mAbs are novel and excellent candidates for further drug development and use in clinical practice.IMPORTANCEIn this study, we identified three human monoclonal antibodies with a high affinity to F1 protein of Yersinia pestis. We discovered that they have relatively lower somatic hypermutations compared with antibodies, m252, αF1Ig2, and αF1Ig8, derived from the naive library reported previously. We also observed that these mAbs share similar binding sites in F1 with some overlapping with αF1Ig8 but distinct from that of αF1Ig2. Furthermore, each of them could provide complete protection for mice against a lethal dose of Yersinia pestis challenge. Our data provided new insights into the anti-F1 Ab repertories and their associated epitopes during vaccination in humans. The findings support the additional novel protective human anti-F1Abs for potential therapeutics against plaque.

An anti-sortilin affibody-peptide fusion inhibits sortilin-mediated progranulin degradation.

Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.

Development of a synthetic library of humanized nanobodies for targeted IL-6 inhibition.

Interleukin-6 (IL-6) is a cytokine that can bind to IL-6 receptor and induce pleiotropic effects. It serves as a critical biomarker, involved in inflammation amplification, tumor progression, and many other disease developments. Nanobodies, featuring small structure and high affinity, are a powerful and versatile tool in medical diagnostics and therapeutics. Here, based on a scaffold optimized for humanization and stability, we developed a synthetic phage display library that rapidly generated high-affinity and humanized nanobodies, negating the need for animal immunization. Using enhanced green fluorescent protein (eGFP) as a benchmark, we demonstrated that the library produced humanized nanobodies with high function and great intracellular stability. The library was then subjected to screening against IL-6. We identified a standout nanobody, NbL3, which exhibited high affinity (22.16 nM) and stability and significantly inhibited IL-6-enhanced migration on the human breast cancer cell MCF-7 at a relatively low concentration. NbL3's strong blocking activity provides a promising therapeutic alternative for the IL-6-targeted intervention strategy, underscoring the broader potential of our synthetic library as a versatile platform for the development of humanized nanobodies against multiple antigens.

Phage display for discovery of anticancer antibodies.

Antibodies and antibody-based immunotherapeutics are the mainstays of cancer immunotherapy. Expanding the repertoire of cancer-specific and cancer-associated epitopes targetable with antibodies represents an important area of research. Phage display is a powerful approach allowing the use of diverse antibody libraries to be screened for binding to a wide range of targets. In this review, we summarize the basics of phage display technology and highlight the advances in anticancer antibody identification and modification via phage display platform. Finally, we describe phage display-derived anticancer monoclonal antibodies that have been approved to date or are in clinical development.

Engineering of pH-dependent antigen binding properties for toxin-targeting IgG1 antibodies using light-chain shuffling.

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.

Construction and validation of a synthetic phage-displayed nanobody library.

Nanobodies derived from camelids and sharks offer unique advantages in therapeutic applications due to their ability to bind to epitopes that were previously inaccessible. Traditional methods of nanobody development face challenges such as ethical concerns and antigen toxicity. Our study presents a synthetic, phagedisplayed nanobody library using trinucleotide-directed mutagenesis technology, which allows precise amino acid composition in complementarity-determining regions (CDRs), with a focus on CDR3 diversity. This approach avoids common problems such as frameshift mutations and stop codon insertions associated with other synthetic antibody library construction methods. By analyzing FDA-approved nanobodies and Protein Data Bank sequences, we designed sub-libraries with different CDR3 lengths and introduced amino acid substitutions to improve solubility. The validation of our library through the successful isolation of nanobodies against targets such as PD-1, ATXN1 and STAT3 demonstrates a versatile and ethical platform for the development of high specificity and affinity nanobodies and represents a significant advance in biotechnology.