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Fulltext Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells

Zuhaida AA, Ali AM, Tamilselvan S, Alitheen NB, Hamid M, Noor AM, et al.

Genet. Mol. Res., 2013;12(4):5547-59.
PMID: 24301925 DOI: 10.4238/2013.November.18.5

A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications.

Matched MeSH terms: Single-Chain Antibodies/immunology*
The design of target specific antibodies (scFv) by applying de novo workflow: Case study on BmR1 antigen from Brugia malayi

Khor BY, Lim TS, Noordin R, Choong YS

J Mol Graph Model, 2017 09;76:543-550.
PMID: 28811153 DOI: 10.1016/j.jmgm.2017.07.004

De novo approach was applied to design single chain fragment variable (scFv) for BmR1, a recombinant antigen from Bm17DIII gene which is the primary antigen used for the detection of anti-BmR1 IgG4 antibodies in the diagnostic of lymphatic filariasis. Three epitopes of the BmR1 was previously predicted form an ab initio derived three-dimensional structure. A collection of energetically favourable conformations was generated via hot-spot-centric approach. This resulted in a set of three different scFv scaffolds used to compute the high shape complementary conformations via dock-and-design approach with the predicted epitopes of BmR1. A total of 4227 scFv designs were generated where 200 scFv designs produced binding energies of less than -20 R.E.U with shape complementarity higher than 0.5. We further selected the design with at least one hydrogen bond and one salt bridge with the epitope, thus resulted in a total of 10, 1 and 19 sFv designs for epitope 1, 2 and 3, respectively. The results thus showed that de novo design can be an alternative approach to yield high affinity in silico scFv designs as a starting point for antibody or specific binder discovery processes.

Matched MeSH terms: Single-Chain Antibodies/immunology
Generation of a naïve human single chain variable fragment (scFv) library for the identification of monoclonal scFv against Salmonella Typhi Hemolysin E antigen

Lim BN, Chin CF, Choong YS, Ismail A, Lim TS

Toxicon, 2016 Jul;117:94-101.
PMID: 27090555 DOI: 10.1016/j.toxicon.2016.04.032

Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets.

Matched MeSH terms: Single-Chain Antibodies/immunology*
Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections

Hamidon NH, Suraiya S, Sarmiento ME, Acosta A, Norazmi MN, Lim TS

Appl Biochem Biotechnol, 2018 Mar;184(3):852-868.
PMID: 28884285 DOI: 10.1007/s12010-017-2582-5

B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 109 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

Matched MeSH terms: Single-Chain Antibodies/immunology*
Mass Spectrometry Immuno Assay (MSIA™) Streptavidin Disposable Automation Research Tips (D.A.R.T's®) Antibody Phage Display Biopanning

Chin CF, Choong YS, Lim TS

Methods Mol Biol, 2018;1701:285-299.
PMID: 29116511 DOI: 10.1007/978-1-4939-7447-4_15

Antibody phage display has been widely established as the method of choice to generate monoclonal antibodies with various efficacies post hybridoma technology. This technique is a popular method which takes precedence over ease of methodology, time- and cost-savings with comparable outcomes to conventional methods. Phage display technology manipulates the genome of M13 bacteriophage to display large diverse collection of antibodies that is capable of binding to various targets (nucleic acids, peptides, proteins, and carbohydrates). This subsequently leads to the discovery of target-related antibody binders. There have been several different approaches adapted for antibody phage display over the years. This chapter focuses on the semi-automated phage display antibody biopanning method utilizing the MSIA™ streptavidin D.A.R.T's® system. The system employs the use of electronic multichannel pipettes with predefined programs to carry out the panning process. The method should also be adaptable to larger liquid handling instrumentations for higher throughput.

Matched MeSH terms: Single-Chain Antibodies/immunology
Fulltext Selection of phage-displayed human antibody fragments specific for CD1b presenting the Mycobacterium tuberculosis glycolipid Ac2SGL

Camacho F, Sarmiento ME, Reyes F, Kim L, Huggett J, Lepore M, et al.

Int J Mycobacteriol, 2016 06;5(2):120-7.
PMID: 27242221 DOI: 10.1016/j.ijmyco.2015.12.002

OBJECTIVE/BACKGROUND: The development of new tools capable of targeting Mycobacterium tuberculosis (Mtb)-infected cells have potential applications in diagnosis, treatment, and prevention of tuberculosis. In Mtb-infected cells, CD1b molecules present Mtb lipids to the immune system (Mtb lipid-CD1b complexes). Because of the lack of CD1b polymorphism, specific Mtb lipid-CD1b complexes could be considered as universal Mtb infection markers. 2-Stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-α-α'-d-trehalose (Ac2SGL) is specific for Mtb, and is not present in other mycobacterial species. The CD1b-Ac2SGL complexes are expressed on the surface of human cells infected with Mtb. The aim of this study was to generate ligands capable of binding these CD1b-Ac2SGL complexes.
METHODS: A synthetic human scFv phage antibody library was used to select phage-displayed antibody fragments that recognized CD1b-Ac2SGL using CD1b-transfected THP-1 cells loaded with Ac2SGL.
RESULTS: One clone, D11-a single, light-variable domain (kappa) antibody (dAbκ11)-showed high relative binding to the Ac2SGL-CD1b complex.
CONCLUSION: A ligand recognizing the Ac2SGL-CD1b complex was obtained, which is a potential candidate to be further tested for diagnostic and therapeutic applications.

Matched MeSH terms: Single-Chain Antibodies/immunology
Development and structural characterisation of human scFv targeting MDM2 spliced variant MDM215kDa

Lim CC, Chan SK, Lim YY, Ishikawa Y, Choong YS, Nagaoka Y, et al.

Mol Immunol, 2021 07;135:191-203.
PMID: 33930714 DOI: 10.1016/j.molimm.2021.04.016

The murine double minute 2 (MDM2) protein is a major negative regulator of the tumour suppressor protein p53. Under normal conditions, MDM2 constantly binds to p53 transactivation domain and/or ubiquinates p53 via its role as E3 ubiquitin ligase to promote p53 degradation as well as nuclear export to maintain p53 levels in cells. Meanwhile, amplification of MDM2 and appearance of MDM2 spliced variants occur in many tumours and normal tissues making it a prognostic indicator for human cancers. The mutation or deletion of p53 protein in half of human cancers inactivates its tumour suppressor activity. However, cancers with wild type p53 have its function effectively inhibited through direct interaction with MDM2 oncoprotein. Here, we described the construction of a MDM2 spliced variant (rMDM215kDa) consisting of SWIB/MDM2 domain and its central region for antibody generation. Biopanning with a human naïve scFv library generated four scFv clones specific to rMDM215kDa. Additionally, the selected scFv clones were able to bind to the recombinant full length MDM2 (rMDM2-FL). Computational prediction showed that the selected scFv clones potentially bind to exon 7-8 of MDM2 while leaving the MDM2/SWIB domain free for p53 interaction. The developed antibodies exhibit good specificity can be further investigated for downstream biomedical and research applications.

Matched MeSH terms: Single-Chain Antibodies/immunology*
Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library

Rahumatullah A, Ahmad A, Noordin R, Lim TS

Mol Immunol, 2015 Oct;67(2 Pt B):512-23.
PMID: 26277276 DOI: 10.1016/j.molimm.2015.07.040

Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.

Matched MeSH terms: Single-Chain Antibodies/immunology*
Fulltext Antibody-Based Protective Immunity against Helminth Infections: Antibody Phage Display Derived Antibodies against BmR1 Antigen

Rahumatullah A, Abdul Karim IZ, Noordin R, Lim TS

Int J Mol Sci, 2017 Nov 22;18(11).
PMID: 29165352 DOI: 10.3390/ijms18112376

Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv) clones against a specific filarial antigen (BmR1). The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications.

Matched MeSH terms: Single-Chain Antibodies/immunology
Fulltext Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies

Lim CC, Woo PCY, Lim TS

Sci Rep, 2019 Apr 15;9(1):6088.
PMID: 30988390 DOI: 10.1038/s41598-019-42628-6

Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.

Matched MeSH terms: Single-Chain Antibodies/immunology
Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology

Mohd Ali MR, Sum JS, Aminuddin Baki NN, Choong YS, Nor Amdan NA, Amran F, et al.

Int J Biol Macromol, 2021 Jan 31;168:289-300.
PMID: 33310091 DOI: 10.1016/j.ijbiomac.2020.12.062

Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually varied, allowing it to be mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of a specific agent in clinical samples is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody against rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.

Matched MeSH terms: Single-Chain Antibodies/immunology
Fulltext Broad specificity of immune helminth scFv library to identify monoclonal antibodies targeting Strongyloides

Rahumatullah A, Balachandra D, Noordin R, Baharudeen Z, Lim YY, Choong YS, et al.

Sci Rep, 2021 01 28;11(1):2502.
PMID: 33510342 DOI: 10.1038/s41598-021-82125-3

Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.

Matched MeSH terms: Single-Chain Antibodies/immunology*
Fulltext Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

Munisvaradass R, Kumar S, Govindasamy C, Alnumair KS, Mok PL

Int J Mol Sci, 2017 Sep 08;18(9).
PMID: 28885562 DOI: 10.3390/ijms18091797

Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non-transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours.

Matched MeSH terms: Single-Chain Antibodies/immunology

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