Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy

Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.

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Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries

International Journal of Molecular Sciences ◽

10.3390/ijms21176354 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6354

Author(s):

Kyungjae Kang ◽

Kicheon Kim ◽

Se-Ra Lee ◽

Yoonji Kim ◽

Joo Eon Lee ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Phage Display ◽

Cancer Therapeutics ◽

Cell Types ◽

Migration Assay ◽

Synthetic Antibody ◽

Phage Display Libraries ◽

Anti Cancer

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.

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Peptides derived from phage display libraries as potential neutralizers of Shiga toxin-induced cytotoxicity in vitro and in vivo

Journal of Applied Microbiology ◽

10.1111/jam.12451 ◽

2014 ◽

Vol 116 (5) ◽

pp. 1322-1333 ◽

Cited By ~ 5

Author(s):

R.A. Bernedo-Navarro ◽

M.M. Miyachiro ◽

M.J. da Silva ◽

C.F. Reis ◽

R.A. Conceição ◽

...

Keyword(s):

Phage Display ◽

Shiga Toxin ◽

Phage Display Libraries

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Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants

10.1101/2022.01.12.22269023 ◽

2022 ◽

Author(s):

Zhaochun Chen ◽

Peng Zhang ◽

Yumiko Matsuoka ◽

Yaroslav Tsybovsky ◽

Kamille West ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Structural Basis ◽

Hamster Model ◽

Golden Syrian Hamster ◽

Phage Display Libraries ◽

Antibody Phage ◽

Antibody Phage Display ◽

Early Isolate

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity1. This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies1,2. Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe3. Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

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Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

Journal of Immunological Methods ◽

10.1016/s0022-1759(03)00070-x ◽

2003 ◽

Vol 278 (1-2) ◽

pp. 271-281 ◽

Cited By ~ 19

Author(s):

Marja-Leena Laukkanen ◽

Soili Mäkinen-Kiljunen ◽

Kirsi Isoherranen ◽

Tari Haahtela ◽

Hans Söderlund ◽

...

Keyword(s):

Phage Display ◽

Single Chain ◽

Single Chain Antibody ◽

Ige Antibodies ◽

Phage Display Libraries ◽

Antibody Phage ◽

Antibody Phage Display

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Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes

Methods in Molecular Biology - Phage Display ◽

10.1007/978-1-4939-7447-4_4 ◽

2017 ◽

pp. 61-82 ◽

Cited By ~ 1

Author(s):

Nam-Kyung Lee ◽

Scott Bidlingmaier ◽

Yang Su ◽

Bin Liu

Keyword(s):

Phage Display ◽

Light Chain ◽

Chain Gene ◽

Human Antibody ◽

Modular Construction ◽

Light Chain Gene ◽

Phage Display Libraries ◽

Antibody Phage ◽

Variable Heavy ◽

Antibody Phage Display

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Neutralizing Antibodies of Botulinum Neurotoxin Serotype A Screened from a Fully Synthetic Human Antibody Phage Display Library

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109343206 ◽

2009 ◽

Vol 14 (8) ◽

pp. 991-998 ◽

Cited By ~ 18

Author(s):

Rui Yu ◽

Shuang Wang ◽

Yun-zhou Yu ◽

Wei-shi Du ◽

Fang Yang ◽

...

Keyword(s):

Phage Display ◽

Neutralizing Antibodies ◽

Botulinum Neurotoxin ◽

Phage Display Library ◽

Human Antibody ◽

Serotype A ◽

Antibody Phage ◽

Antibody Phage Display ◽

Botulinum Neurotoxin Serotype A

The botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous protein substances known. The neutralizing antibodies against botulinum neurotoxin can effectively prevent and cure the toxicosis. Using purified Hc fragments of botulinum neurotoxin serotype A (BoNT/A-Hc) as antigen, 2 specific neutralizing antibodies mapping different epitopes were selected from a fully synthetic human antibody library. The 2 antibodies can effectively inhibit the binding between BoNT/A-Hc and differentiated PC-12 cells in vitro, and the neutralization was evaluated in vivo. Although no single mAb completely protected mice from toxin, they both could prolong time to death when challenged with 20 LD 50s (50% lethal doses) of BoNT/A. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, suggesting their high neutralizing potency in vivo . The results would lead to further production of neutralizing antibody drugs against BoNT/A. It also proved that it was a quick method to obtain human therapeutic antibodies by selecting from the fully synthetic human antibody phage display library. ( Journal of Biomolecular Screening 2009:991-998)

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Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus

Journal of Virology ◽

10.1128/jvi.79.3.1635-1644.2005 ◽

2005 ◽

Vol 79 (3) ◽

pp. 1635-1644 ◽

Cited By ~ 81

Author(s):

Edward N. van den Brink ◽

Jan ter Meulen ◽

Freek Cox ◽

Mandy A. C. Jongeneelen ◽

Alexandra Thijsse ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Vero Cells ◽

Human Monoclonal Antibodies ◽

Human Mabs ◽

N Protein ◽

Antibody Phage ◽

Antibody Phage Display

ABSTRACT Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

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Neutralizing Human Fab Fragments against Measles Virus Recovered by Phage Display

Journal of Virology ◽

10.1128/jvi.76.1.251-258.2002 ◽

2002 ◽

Vol 76 (1) ◽

pp. 251-258 ◽

Cited By ~ 25

Author(s):

Cristina de Carvalho Nicacio ◽

R. Anthony Williamson ◽

Paul W. H. I. Parren ◽

Åke Lundkvist ◽

Dennis R. Burton ◽

...

Keyword(s):

Phage Display ◽

Measles Virus ◽

Protein Band ◽

Enzyme Linked Immunosorbent Assay ◽

Neutralization Capacity ◽

N Protein ◽

Fab Fragments ◽

Phage Display Libraries ◽

Antibody Phage ◽

Antibody Phage Display

ABSTRACT Five human recombinant Fab fragments (Fabs) specific for measles virus (MV) proteins were isolated from three antibody phage display libraries generated from RNAs derived from bone marrow or splenic lymphocytes from three MV-immune individuals. All Fabs reacted in an enzyme-linked immunosorbent assay with MV antigens. In radioimmunoprecipitation assays two of the Fabs, MV12 and MT14, precipitated an ⊘80-kDa protein band corresponding to the hemagglutinin (H) protein from MV-infected Vero cell cultures, while two other Fabs, MT64 and GL29, precipitated an ⊘60-kDa protein corresponding the nucleocapsid (N) protein. In competition studies with MV fusion, H- and N protein-specific monoclonal antibodies (MAbs), the H-specific Fabs predominantly blocked the binding of H-specific MAbs, while the N-specific Fabs blocked MAbs to N. In addition, N-specific Fabs bound to denatured MV N protein in Western blotting. The specificity of the fifth Fab, MV4, could not be determined. By plaque reduction assays, three of the five Fabs, MV4, MV12, and MT14, exhibited neutralizing activity (80% cutoff) against MV (LEC-KI strain) at concentrations ranging between ≈2 and 7 μg ml−1. Neutralization capacity against MV strains Edmonston and Schwarz was also detected, albeit at somewhat higher Fab concentrations. In conclusion, three neutralizing Fabs were isolated, two of them reactive against the H glycoprotein of MV and another reactive against an undefined epitope. This is the first study in which MV-neutralizing human recombinant Fab antibodies have been isolated from phage display libraries.

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