Abstract:The development of single‐chain variable fragments (scFvs) as therapeutic agents has the potential to reduce the high cost of antibody production, but the development process often impairs scFv functions such as binding affinity and pharmacokinetics. Multimerization is one strategy for recovering or enhancing these lost functions. Previously, we constructed several antiepidermal growth factor receptor (
EGFR
) scFv multimers by modifying linker length and domain order. Antitumor effects … Show more
“…We propose that the method developed by us may be useful for engineering VL fragments for affinity maturation. Furthermore, we previously confirmed cytotoxic enhancement of Ex3 bsDb for cancer growth inhibition by integrating high-affinity VH mutants 19 , and have reported several recombinant cancer therapeutic antibodies based on h528 27 , 28 , 39 , 40 . These cytotoxic enhancements can also be made by integrating the isolated VL mutants into VH mutants and may result in the development of more desirable molecules.…”
Section: Discussionmentioning
confidence: 62%
“…Rise in the binding affinity was observed in both the mutants, and 2L6 exhibited an affinity almost equivalent to that of parental m528 Fv. By screening h528 VL mutants, further enhancement of h528 affinity was accomplished, and we expect the resultant h528 Fv mutants to further improve our engineered antibodies based on h528 Fv for cancer therapy 19 , 27 , 28 . Figure 4 Titration calorimetry of the interaction between h528 Fv mutants and sEGFR.…”
Section: Resultsmentioning
confidence: 99%
“…Using this novel Fv-added OS method, we successfully isolated h528 VL mutants with high affinity. This method may also be useful for engineering antibody VL fragments and integrating isolated high-affinity VL mutants into engineered antibodies previously constructed by us based on h528 Fv 19 , 27 , 28 for increasing their affinity and tumour-inhibitory effects.…”
Affinity maturation is one of the cardinal strategies for improving antibody function using in vitro evolutionary methods; one such well-established method is phage display. To minimise gene deletion, we previously developed an open sandwich (OS) method wherein selection was performed using only phage-displaying VH fragments after mixing with soluble VL fragments. The decrease in anti-EGFR antibody 528 affinity through humanization was successfully recovered by selecting VH mutants using this OS method. However, the affinity was not similar to that of parental 528. For further affinity maturation, we aimed to isolate VL mutants that act in synergy with VH mutants. However, the OS method could not be applied for selecting VL fragments because the preparation of soluble VH fragments was hampered by their instability and insolubility. Therefore, we initially designed a modified OS method based on domain-swapping of VH fragments, from added soluble Fv fragments to phage-displaying VL fragments. Using this novel Fv-added OS selection method, we successfully isolated VL mutants, and one of the Fv comprising VH and VL mutants showed affinity almost equivalent to that of parental 528. This method is applicable for engineering other VL fragments for affinity maturation.
“…We propose that the method developed by us may be useful for engineering VL fragments for affinity maturation. Furthermore, we previously confirmed cytotoxic enhancement of Ex3 bsDb for cancer growth inhibition by integrating high-affinity VH mutants 19 , and have reported several recombinant cancer therapeutic antibodies based on h528 27 , 28 , 39 , 40 . These cytotoxic enhancements can also be made by integrating the isolated VL mutants into VH mutants and may result in the development of more desirable molecules.…”
Section: Discussionmentioning
confidence: 62%
“…Rise in the binding affinity was observed in both the mutants, and 2L6 exhibited an affinity almost equivalent to that of parental m528 Fv. By screening h528 VL mutants, further enhancement of h528 affinity was accomplished, and we expect the resultant h528 Fv mutants to further improve our engineered antibodies based on h528 Fv for cancer therapy 19 , 27 , 28 . Figure 4 Titration calorimetry of the interaction between h528 Fv mutants and sEGFR.…”
Section: Resultsmentioning
confidence: 99%
“…Using this novel Fv-added OS method, we successfully isolated h528 VL mutants with high affinity. This method may also be useful for engineering antibody VL fragments and integrating isolated high-affinity VL mutants into engineered antibodies previously constructed by us based on h528 Fv 19 , 27 , 28 for increasing their affinity and tumour-inhibitory effects.…”
Affinity maturation is one of the cardinal strategies for improving antibody function using in vitro evolutionary methods; one such well-established method is phage display. To minimise gene deletion, we previously developed an open sandwich (OS) method wherein selection was performed using only phage-displaying VH fragments after mixing with soluble VL fragments. The decrease in anti-EGFR antibody 528 affinity through humanization was successfully recovered by selecting VH mutants using this OS method. However, the affinity was not similar to that of parental 528. For further affinity maturation, we aimed to isolate VL mutants that act in synergy with VH mutants. However, the OS method could not be applied for selecting VL fragments because the preparation of soluble VH fragments was hampered by their instability and insolubility. Therefore, we initially designed a modified OS method based on domain-swapping of VH fragments, from added soluble Fv fragments to phage-displaying VL fragments. Using this novel Fv-added OS selection method, we successfully isolated VL mutants, and one of the Fv comprising VH and VL mutants showed affinity almost equivalent to that of parental 528. This method is applicable for engineering other VL fragments for affinity maturation.
“…Although scFv multimerization depends on both structural and environmental factors, the candidate drug was less costly while retaining a high inhibitory effect and long in vivo half-life. 68 Although most of current scFv immunotherapies depend on fusing with separate biomaterials, constant attention to its development is highly recommended for its potential extensions.…”
Section: Extensions In Therapeutic Antibodymentioning
confidence: 99%
“…Adverse effects were absent, and sternal metastatic regions were clearly identified for 24 h after treatment in both single-photon emission computed tomography (SPECT) and computed tomography (CT) imaging. 82 A similar phase I trial was conducted for 68 Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid ( 68 Ga-NOTA)-anti-HER2 VHH1, which additionally underwent positron emission tomography (PET) imaging. Fast blood clearance was observed, and the tracer accumulation was substantial for identifying most sites of disease.…”
Immunotherapy has been well regarded as one of the safer and antigen-specific anti-cancer treatments compared to first-generation chemotherapy. Since Coley's discovery, researchers focused on engineering novel antibody-based therapies. Including artificial and modified antibodies, such as antibody fragments, antibody-drug conjugates, and synthetic mimetics, the variety of immunotherapy has been rapidly expanding in the last few decades. Genetic and chemical modifications to monoclonal antibody have been brought into academia, in vivo trials, and clinical applications. Here, we have looked around antibodies overall. First, we elucidate the antibody structure and its cytotoxicity mechanisms. Second, types of therapeutic antibodies are presented. Additionally, there is a summarized list of US Food and Drug Administration (FDA)-approved therapeutic antibodies and recent clinical trials. This review provides a comprehensive overview of both the general function of therapeutic antibodies and a few main variations in development, including recent advent with the proposed mechanism of actions, and we introduce types of therapeutic antibodies, clinical trials, and approved commercial immunotherapeutic drugs.
STRUCTURES AND FUNCTIONS OF THERAPEUTIC ANTIBODY
Structure of therapeutic antibodyIgs have five major classes where each class has a unique sequence of heavy chain (HC) constant regions: IgA, IgD, IgE, IgG, and IgM. 10 IgG, which has a g HC, comprises 80% of total antibodies in serum,
Single-chain variable fragment (scFv) antibodies as therapeutic agents have the potential to reduce the production cost and immunogenicity relative to monoclonal antibodies, but their monovalency and lack of a fragment crystallizable region can lead to reduced function. Multimerization is one strategy for recovering the function; however, their application is limited by the production of multimeric proteins. In our previous study, an anti-lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) scFv showed potential use in diagnosis and therapy of atherosclerotic diseases, but is limited by its inherent low antigen-binding activity. In this study, to improve the efficacy of the anti-LOX-1 scFv, we constructed the anti-LOX-1 scFv multimers by modifying the linker length between the variable domains of the scFv or by fusing the scFv with self-merization domains and expressed these scFv multimers in Brevibacillus choshinensis hosts. After optimization, all of the scFv multimers obtained efficient secretion expression. Compared with the scFv monomer, the multimers that are successfully fractionated displayed increased neutralization activity and showed elevated antigen-binding avidity, especially the tetramer, which improved the antigen avidity by two orders of magnitude. Moreover, the scFv dimer and the tetramer both displayed better stability and longer half-life in serum, which can be attractive candidates for the next-generation anti-LOX-1 therapeutic antibody.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
