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1.
Front Immunol ; 14: 1089395, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37180155

RESUMO

Background: Monoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications. Methods: The library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to ß-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays. Results: We have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation. Conclusion: The promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.


Assuntos
Bacteriófagos , Anticorpos de Cadeia Única , Humanos , Biblioteca de Peptídeos , Receptor Celular 2 do Vírus da Hepatite A , Regiões Determinantes de Complementaridade/química , Anticorpos Monoclonais , Anticorpos de Cadeia Única/genética , Anticorpos Neutralizantes
2.
Methods Mol Biol ; 2552: 437-445, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346607

RESUMO

To ensure the functionalities of the antibodies in phage-displayed synthetic antibody libraries, we use computational method to evaluate the designs of the antibody libraries. The computational methodologies developed in our lab for designing antibody library provide rich information on the function of the designed antibody sequences-adequate antibody designs for a specific antigen type should have predicted paratopes for the antigen type. This computational assessment of the designed antibody sequences helps eliminate non-functional designs before proceeding to construct the library designs in the wet lab. As such, only reasonable antibody designs are constructed for antibody discoveries.


Assuntos
Anticorpos , Biblioteca de Peptídeos , Sítios de Ligação de Anticorpos , Antígenos
3.
Antib Ther ; 4(2): 101-108, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34195544

RESUMO

Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. Recently, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Using VH3-23 as a model framework, we observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Our observations shed light on the design of next-generation synthetic diversity with improved probability of success.

4.
Methods Protoc ; 2(1)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-31164599

RESUMO

Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length.

5.
MAbs ; 11(2): 373-387, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30526270

RESUMO

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.


Assuntos
Aprendizado de Máquina , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/química , Afinidade de Anticorpos , Especificidade de Anticorpos , Humanos , Biblioteca de Peptídeos , Relação Estrutura-Atividade
6.
MAbs ; 11(1): 153-165, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30365359

RESUMO

HER2-ECD (human epidermal growth factor receptor 2 - extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies' propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.


Assuntos
Aminobenzoatos/farmacologia , Imunoconjugados/farmacologia , Terapia de Alvo Molecular/métodos , Oligopeptídeos/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Neoplasias Gástricas/patologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Microbiol Biotechnol ; 28(8): 1376-1383, 2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-30301315

RESUMO

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity (KD) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Biblioteca de Peptídeos , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Bacteriófagos/genética , Genótipo , Células HEK293 , Células Hep G2 , Anticorpos Anti-Hepatite B/imunologia , Anticorpos Anti-Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Testes de Neutralização , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo
8.
Methods Mol Biol ; 1701: 147-167, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29116504

RESUMO

Besides classical antibodies with the composition of heavy and light chains, sharks produce a unique heavy chain only isotype, termed Immunoglobulin New Antigen Receptor (IgNAR), in which antigen binding is solely mediated by a single domain, referred to as vNAR. Owing to their high affinity and specificity combined with their small size and high stability, vNAR domains emerged as promising target-binding scaffolds that can be tailor-made for biotechnological and biomedical applications. Herein, we describe protocols for the construction of semi-synthetic, CDR3-randomized vNAR libraries for the isolation of target-specific antibodies using yeast surface display or phage display as platform technology. Additionally, we provide information for affinity maturation of target-specific molecules through CDR1 diversification and sublibrary establishment.


Assuntos
Anticorpos Monoclonais/genética , Proteínas de Peixes/genética , Biblioteca Gênica , Receptores de Antígenos/genética , Tubarões/genética , Animais , Anticorpos Monoclonais/imunologia , Proteínas de Peixes/imunologia , Receptores de Antígenos/imunologia , Tubarões/imunologia
9.
MAbs ; 10(2): 256-268, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29227213

RESUMO

The commercial success of bispecific antibodies generally has been hindered by the complexities associated with generating appropriate molecules for both research scale and large scale manufacturing purposes. Bispecific IgG (BsIgG) based on two antibodies that use an identical common light chain can be combined with a minimal set of Fc mutations to drive heavy chain heterodimerization in order to address these challenges. However, the facile generation of common light chain antibodies with properties similar to traditional monoclonal antibodies has not been demonstrated and they have only been used sparingly. Here, we describe the design of a synthetic human antibody library based on common light chains to generate antibodies with biochemical and biophysical properties that are indistinguishable to traditional therapeutic monoclonal antibodies. We used this library to generate diverse panels of well-behaved, high affinity antibodies toward a variety of epitopes across multiple antigens, including mouse 4-1BB, a therapeutically important T cell costimulatory receptor. Over 200 BsIgG toward 4-1BB were generated using an automated purification method we developed that enables milligram-scale production of BsIgG. This approach allowed us to identify antibodies with a wide range of agonistic activity that are being used to further investigate the therapeutic potential of antibodies targeting one or more epitopes of 4-1BB.


Assuntos
Anticorpos Biespecíficos , Cadeias Leves de Imunoglobulina , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Animais , Humanos , Camundongos
10.
Methods Mol Biol ; 1575: 15-29, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28255872

RESUMO

Many large synthetic antibody libraries have been designed, constructed, and successfully generated high-quality antibodies suitable for various demanding applications. While synthetic antibody libraries have many advantages such as optimized framework sequences and a broader sequence landscape than natural antibodies, their sequence diversities typically are generated by random combinatorial synthetic processes which cause the incorporation of many undesired CDR sequences. Here, we describe the construction of a synthetic scFv library using oligonucleotide mixtures that contain predefined, non-combinatorially synthesized CDR sequences. Each CDR is first inserted to a master scFv framework sequence and the resulting single-CDR libraries are subjected to a round of proofread panning. The proofread CDR sequences are assembled to produce the final scFv library with six diversified CDRs.


Assuntos
Regiões Determinantes de Complementaridade/genética , Biblioteca Gênica , Anticorpos de Cadeia Única/genética , Sequência de Aminoácidos , Regiões Determinantes de Complementaridade/imunologia , Genes Sintéticos , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/imunologia
11.
Adv Exp Med Biol ; 1053: 21-34, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29549633

RESUMO

Antibody phage display has become an indispensable tool for the discovery and optimization of target-specific monoclonal antibodies suitable for demanding applications including therapeutic reagents. The in vitro nature of the technology enables the rapid and efficient identification of specific binders, as well as greater control over selection parameters that facilitates the isolation of antibodies with unique, desirable functional characteristics. In this chapter, the technological background and the state of the art in the field of antibody phage display is discussed.


Assuntos
Anticorpos Monoclonais/genética , Bacteriófago M13/genética , Técnicas de Visualização da Superfície Celular , Escherichia coli/virologia , Fragmentos Fab das Imunoglobulinas/genética , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genética , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêutico
12.
Protein Eng Des Sel ; 28(10): 317-25, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26333274

RESUMO

Display technologies such as yeast and phage display offer powerful alternatives to traditional immunization-based antibody discovery, but require conversion of displayed proteins into soluble form prior to downstream characterization. Here we utilize amber suppression to implement a yeast-based switchable display/secretion system that enables the immediate production of soluble, antibody-like reagents at the end of screening efforts. Model selections in the switchable format remain efficient, and library screening in the switchable format yields renewable sources of affinity reagents exhibiting nanomolar binding affinities. These results confirm that this system provides a seamless link between display-based screening and the production and evaluation of soluble forms of candidate binding proteins. Switchable display/secretion libraries provide a cloning-free, accessible approach to affinity reagent generation.


Assuntos
Engenharia de Proteínas/métodos , Leveduras/genética , Linhagem Celular , Clonagem Molecular , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Conformação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
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