FIGURE 3 - available via license: Creative Commons Attribution 4.0 International
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Schematic representation of in vitro Phage Display Technology. (A) In phage display systems, antibody genes are linked to the amino terminus region of the phage minor coat protein pIII, as shown in the phagemid. When expressed, mature phages will incorporate the encoded fusion product, creating a link between antibody genotype and phenotype. (B) In vitro phage display, selection is composed of several steps: coating of the antigen or preparation of the cells; incubation of phage repertoire with antigen; washing to remove non-specific phages; and elution and reamplification of antigen-specific phages. The stringency of selection could be increased by the increase in the number of washing steps.
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The discovery of hybridoma technology, described by Kohler and Milstein in 1975, and the resulting ability to generate monoclonal antibodies (mAbs) initiated a new era in antibody research and clinical development. However, limitations of the hybridoma technology as a routine antibody generation method in conjunction with high immunogenicity respon...
Citations
... This chimerization process seeks to retain the specificity and affinity of human antibodies while benefiting from the stability and expression advantages offered by non-human constant regions. Chimeric libraries can also be generated by combining variable domains from different antibodies, allowing for the exploration of diverse binding specificities within a single library [30]. ...
... In contrast, immune libraries derive from B-cell antibody repertoires of immunized or immune donors, exhibiting a predisposition to a limited panel of antigens and generally being smaller in size. While adept at addressing specific antigens, immune libraries are less suited for identifying antibody fragments against a broad spectrum of antigens, particularly self-antigens [30]. Synthetic libraries leverage computational design and gene synthesis, affording precise control over the composition of complementarity-determining regions (CDRs) [28]. ...
Thirty-four years ago, the groundbreaking work of John McCafferty and Sir Gregory Winter in developing phage display technology revolutionized the discovery of human antibodies, paving the way for diverse applications. Since then, numerous phage-derived antibodies have been successfully developed and advanced into clinical studies, resulting in the approval of more than a dozen therapeutic antibodies. These antibodies have demonstrated efficacy across a spectrum of medical conditions, ranging from autoimmune diseases to various cancers. In this article, we provide an in-depth review of the development of phage display libraries as powerful platforms for therapeutic antibody discovery, elucidating the intricate procedures involved in antibody development. Additionally, we conduct a review of the current ntibody drugs for cancer treatment that have been developed using the phage display platform. Furthermore, we discuss the challenges inherent in this technology, offering insights into potential solutions to enhance crucial steps and facilitate more efficient drug discovery in the field of phage display technology.
... This resulted in the fusion of foreign peptides with the G3P coat protein of the M13 phage [3]. Approximately five years later, McCafferty et al. described the initial application of antibody phage display selection [18]. They successfully fused genes encoding an entire antibody binding domain, specifically single-chain variable fragments (scFvs), to gene II of the M13 phage [13]. ...
... Phage display libraries can obtain gene repertoires from animals that are either naïve or immunized, or these libraries can be synthetically constructed by incorporating randomized complementaritydetermining region (CDR) sequences into fixed frameworks [18]. ...
... To facilitate the display of the fragment on the outer surface of a bacteriophage, the recombinant scFv is genetically fused to a specific protein of the bacteriophage [14][15][16]. The isolation of scFvs with the desired affinity and specificity can be achieved effectively through phage panning, which involves the selection of phages that exhibit adherence to the target [15][16][17][18]. ...
Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell–cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte’s mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.
... As of August 2022, 14 FDA/EMA-approved therapeutic antibodies have been developed using phage display, including the world's bestselling antibody, adalimumab. 24 The technology provides a rapid method to isolate numerous unique scFvs that can be screened for desired functional and developability characteristics to identify lead candidates to move to a clinical setting. Furthermore, as the phenotype and genotype of each scFv are linked within the phage, the VH and VL nucleotide sequences encoding the desired scFv can be rapidly determined and used to generate alternative antibody-based formats including bi-specific T cell engagers, bi-specific antibodies, and chimeric antigen receptor constructs that can be tested. ...
PD-1 checkpoint inhibitors have revolutionized the treatment of patients with different cancer histologies including melanoma, renal cell carcinoma, and non-small cell lung carcinoma. However, only a subset of patients show a dramatic clinical response to treatment. Despite intense biomarker discovery efforts, no single robust, prognostic correlation has emerged as a valid outcome predictor. Immune competent, pet dogs develop spontaneous tumors that share similar features to human cancers including chromosome aberrations, molecular subtypes, immune signatures, tumor heterogeneity, metastatic behavior, and chemotherapeutic response. As such, they represent a valuable parallel patient population in which to investigate predictive biomarkers of checkpoint inhibition. However, the lack of a validated, non-immunogenic, canine anti-PD-1 antibody for pre-clinical use hinders this comparative approach and prevents potential clinical benefits of PD-1 blockade being realized in the veterinary clinic. To address this, fully canine single-chain variable fragments (scFvs) that bind canine (c)PD-1 were isolated from a comprehensive canine scFv phage display library. Lead candidates were identified that bound with high affinity to cPD-1 and inhibited its interaction with canine PD-L1 (cPD-L1). The lead scFv candidate re-formatted into a fully canine IgGD reversed the inhibitory effects of cPD-1:cPD-L1 interaction on canine chimeric antigen receptor (CAR) T cell function. In vivo administration showed no toxicity and revealed favorable pharmacokinetics for a reasonable dosing schedule. These results pave the way for clinical trials with anti-cPD-1 in canine cancer patients to investigate predictive biomarkers and combination regimens to inform human clinical trials and bring a promising checkpoint inhibitor into the veterinary armamentarium.
... Phage technology is not only widely employed in vitro screening, but also utilized for in vivo screening of peptides and antibodies. [132] Antibodies capable of penetrating the blood-brain barrier were discovered, with an accumulation as high as 0.82 ± 0.05% I.A./g found within the brain. [133] In addition to phage technology, Yan et al. reported a method of localized instillation for the in vivo screening of targeting peptides using the OBOC technique. ...
Materialomics integrates experiment, theory, and computation in a high‐throughput manner, and has changed the paradigm for the research and development of new functional materials. Recently, with the rapid development of high‐throughput characterization and machine‐learning technologies, the establishment of biomaterialomics that tackles complex physiological behaviors has become accessible. Breakthroughs in the clinical translation of nanoparticle‐based therapeutics and vaccines have been observed. Herein, recent advances in biomaterials, including polymers, lipid‐like materials, and peptides/proteins, discovered through high‐throughput screening or machine learning‐assisted methods, are summarized. The molecular design of structure‐diversified libraries; high‐throughput characterization, screening, and preparation; and, their applications in drug delivery and clinical translation are discussed in detail. Furthermore, the prospects and main challenges in future biomaterialomics and high‐throughput screening development are highlighted.
... After a period of time, the unbound phages are eluted and those bound to the target organ are collected and amplified by the recipient bacterium ER2738 for the next round of screening. The desired organs/tissues are then collected, homogenized, and the phages are extracted for sequencing (Muttenthaler et al., 2021;André et al., 2022; Figure 4B). This process allows for the identification of specific peptides that target particular organs or tissues in vivo. ...
... The host immune system may recognize and clear the phage particles, limiting the amount of time they can interact with the target tissues. Additionally, one limitation of traditional animal models is the potential differences in peptide binding between species, which can limit the translational potential of identified peptides (André et al., 2022). Ex vivo screening is particularly useful for selecting rare cells in heterogeneous populations (Saw and Song, 2019). ...
Pancreatic cancer is a devastating disease with a high mortality rate and a lack of effective therapies. The challenges associated with early detection and the highly aggressive nature of pancreatic cancer have limited treatment options, underscoring the urgent need for better disease-modifying therapies. Peptide-based biotherapeutics have become an attractive area of research due to their favorable properties such as high selectivity and affinity, chemical modifiability, good tissue permeability, and easy metabolism and excretion. Phage display, a powerful technique for identifying peptides with high affinity and specificity for their target molecules, has emerged as a key tool in the discovery of peptide-based drugs. Phage display technology involves the use of bacteriophages to express peptide libraries, which are then screened against a target of interest to identify peptides with desired properties. This approach has shown great promise in cancer diagnosis and treatment, with potential applications in targeting cancer cells and developing new therapies. In this comprehensive review, we provide an overview of the basic biology of phage vectors, the principles of phage library construction, and various methods for binding affinity assessment. We then describe the applications of phage display in pancreatic cancer therapy, targeted drug delivery, and early detection. Despite its promising potential, there are still challenges to be addressed, such as optimizing the selection process and improving the pharmacokinetic properties of phage-based drugs. Nevertheless, phage display represents a promising approach for the development of novel targeted therapies in pancreatic cancer and other tumors.
... However, developing a strong binding efficacy in the nanomolar range might be possible through the selection of different phage colonies. The selection process involves the screening of a large library of phage-displayed protein variants to identify the strongest binding affinity and optimizing conditions to enhance the binding properties [38]. Taken together, this study demonstrated the characteristics of an scFv against the ECD of the FSHr developed by phage-display technology. ...
FSHr antibodies have been shown to inhibit the differentiation of spermatogonia to primary spermatocytes, resulting in infertility without a pathological effect on reproductive organs. The aim of this study was to develop single-chain variable fragments (scFvs) against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and to evaluate the effects of intratesticular administration of the anti-FSHr scFv on testicular function and testosterone production. A phage clone against the extracellular domain of FSHr selected from a scFv phagemid library was analyzed for binding kinetics by surface plasmon resonance. Using ultrasound guidance, three adult macaques (M. fascicularis) were administered with 1 mL of 0.4 mg/mL anti-FSHr scFv (treatment) and 1 mL sterile phosphate buffer solution (control) into the left and right rete testis, respectively. Testicular appearance and volume, ejaculate quality, and serum testosterone levels were recorded on day 0 (before injection) and on days 7, 28, and 56 (after injection). Testicular tissue biopsies were performed on day 7 and day 56 to quantify the mRNA expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB), and vascular endothelial growth factor A (VEGFA). The results demonstrated that the anti-FSHr scFv molecule was calculated as 27 kDa with a dissociation constant (KD) of 1.03 µM. The volume of the anti-FSHr scFv-injected testicle was reduced on days 28 and 56 compared with day 0 (p < 0.05). Total sperm number was reduced from day 0 (36.4 × 106 cells) to day 56 (1.6 × 106 cells) (p < 0.05). The percentage of sperm motility decreased from day 0 (81.7 ± 1.0%) to day 7 (23.3 ± 1.9%), day 28 (41.7 ± 53.4%), and day 56 (8.3 ± 1.9%) (p < 0.05). Sperm viability on day 0 was 86.8 ± 0.5%, which reduced to 64.2 ± 1.5%, 67.1 ± 2.2%, and 9.3 ± 1.1% on days 7, 28, and 56, respectively (p < 0.05). The expression of ABP and VEGFA on days 7 (14.2- and 3.2-fold) and 56 (5.6- and 5.5-fold) was less in the scFv-treated testicle compared with the controls (p < 0.05). On day 56, the expression of IHBB was less (p < 0.05) in the treated testis (1.3-fold) compared with the controls. Serum testosterone levels were unchanged throughout the study period (p > 0.05). This study characterized the anti-FSHr scFv and demonstrated that treatment with anti-FSHr ameliorates testicular function without altering testosterone levels, offering a potential alternative contraceptive for the long-tailed macaques.
... This technique enables the production of antibodies of a defined specificity in large quantities ex vivo. Alternatively, more efficient methods of identifying mAbs have been explored, including various in vitro display technologies, in vivo phage display, and flow cytometry-based B cell screening and sorting [9][10][11]. The formats of mAbs have been improved over the years, from murine, chimeric, humanized, to fully human mAbs with successively decreasing degrees of immunogenicity in recipients [12]. ...
Harnessing the immune system to combat disease has revolutionized medical treatment. Monoclonal antibodies (mAbs), in particular, have emerged as important immunotherapeutic agents with clinical relevance in treating a wide range of diseases, including allergies, autoimmune diseases, neurodegenerative disorders, cancer, and infectious diseases. These mAbs are developed from naturally occurring antibodies and target specific epitopes of single molecules, minimizing off-target effects. Antibodies can also be designed to target particular pathogens or modulate immune function by activating or suppressing certain pathways. Despite their benefit for patients, the production and administration of monoclonal antibody therapeutics are laborious, costly, and time-consuming. Administration often requires inpatient stays and repeated dosing to maintain therapeutic levels, limiting their use in underserved populations and developing countries. Researchers are developing alternate methods to deliver monoclonal antibodies, including synthetic nucleic acid-based delivery, to overcome these limitations. These methods allow for in vivo production of monoclonal antibodies, which would significantly reduce costs and simplify administration logistics. This review explores new methods for monoclonal antibody delivery, including synthetic nucleic acids, and their potential to increase the accessibility and utility of life-saving treatments for several diseases.
... Here, screening for individual Nb clones that perform well can be completed in parallel with the selection procedure [51]. Andre provided an overview of in vivo phage display methodologies, highlighting them as a promising emerging approach for enhancing antibody targeting and improving the characteristics of drug delivery [62]. Figure 2 provides an overview of the nanobody production procedure. ...
Simple Summary
Targeted alpha therapy (TαT) has revolutionized cancer treatment by delivering high-energy but short-range particles directly to tumor cells. The discovery of single-domain antibodies, or nanobodies, has opened new avenues for TαT. Owing to their small size, nanobodies exhibit excellent binding affinity and specificity, along with significant tumor uptake. Radiolabeled nanobodies offer numerous advantages over traditional TαT delivery vehicles and can be utilized not only for therapeutic purposes but also for cancer imaging. This review will delve into the properties of nanobodies in more detail and highlight recent studies involving nanobody-based TαT.
Abstract
The persistent threat of cancer necessitates the development of improved and more efficient therapeutic strategies that limit damage to healthy tissues. Targeted alpha therapy (TαT), a novel form of radioimmuno-therapy (RIT), utilizes a targeting vehicle, commonly antibodies, to deliver high-energy, but short-range, alpha-emitting particles specifically to cancer cells, thereby reducing toxicity to surrounding normal tissues. Although full-length antibodies are often employed as targeting vehicles for TαT, their high molecular weight and the presence of an Fc-region lead to a long blood half-life, increased bone marrow toxicity, and accumulation in other tissues such as the kidney, liver, and spleen. The discovery of single-domain antibodies (sdAbs), or nanobodies, naturally occurring in camelids and sharks, has introduced a novel antigen-specific vehicle for molecular imaging and TαT. Given that nanobodies are the smallest naturally occurring antigen-binding fragments, they exhibit shorter relative blood half-lives, enhanced tumor uptake, and equivalent or superior binding affinity and specificity. Nanobody technology could provide a viable solution for the off-target toxicity observed with full-length antibody-based TαT. Notably, the pharmacokinetic properties of nanobodies align better with the decay characteristics of many short-lived α-emitting radionuclides. This review aims to encapsulate recent advancements in the use of nanobodies as a vehicle for TαT.
... This enables the reduction of the off-target tissue and protein interactions by eliminating non-specific ligands, enriching the recovery of specific ligands that specifically target the tumor. Despite its advantages, in vivo phage display selection remains relatively unexplored44,45 . To date, few studies on cancer models have been reported.Soendergaard et al. used in vivo phage display selection to identify an ovarian cancer targeting peptide46 . ...
Antibody–drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.
