Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

Wu, Baoping; Xiao, Bing; Wan, Tian-Mo; Zhang, Yali; Zhang, Zhen-Shu; Zhou, Dian-Yuan; Lai, Zhuo-sheng; Gao, Chunfang

doi:10.3748/wjg.v7.i6.811

Cited by 11 publications

(4 citation statements)

References 33 publications

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“…All these families were cloned into pCDisplay3 to ensemble the library. Our library size was compatible with the size reported to other constructed libraries, which range from 10 6 to 10 10 [ 38 , 39 , 40 ]. However, it is very laborious and time-consuming to build large libraries (>10 10 ) due to both the use of a large number of blood samples [ 41 ] and bacteria transformations [ 42 , 43 ].…”

Section: Discussionsupporting

confidence: 70%

Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display

Effer,

Ulloa,

Dappolonnio

et al. 2024

Antibodies

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Gallbladder cancer (GBC) is a very aggressive malignant neoplasm of the biliary tract with a poor prognosis. There are no specific therapies for the treatment of GBC or early diagnosis tools; for this reason, the development of strategies and technologies that facilitate or allow an early diagnosis of GBC continues to be decisive. Phage display is a robust technique used for the production of monoclonal antibodies (mAbs) involving (1) the generation of gene libraries, (2) the screening and selection of isoforms related to an immobilized antigen, and (3) the in vitro maturation of the affinity of the antibody for the antigen. This research aimed to construct a human immune library from PBMCs of GBC patients and the isolation of scFv-phage clones with specificity against the larger extracellular loop belonging to claudin 18.2, which is an important biomarker overexpressed in GBC as well as gastric cancer. The immune-library-denominated GALLBLA1 was constructed from seven GBC patients and has a diversity of 6.12 × 1010 pfu mL−1. After three rounds of panning, we were able to identify clones with specificity against claudin 18.2. GALLBLA1 can contribute to the selection, isolation, and recombinant production of new human mAbs candidates for the treatment of gastrointestinal cancers.

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Section: Discussionsupporting

confidence: 70%

Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display

Effer,

Ulloa,

Dappolonnio

et al. 2024

Antibodies

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show abstract

“…The phage display antibody library can be panned to select and clone antibodies based on their binding specificities. Numerous high affinity antibodies have been obtained using this approach [16][17][18][19][20].…”

mentioning

confidence: 99%

An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells

Wen¹,

Qin²,

Gao³

et al. 2007

Journal of Viral Hepatitis

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Hepatitis B virus surface antigen (HBsAg), a specific antigen on the membrane of hepatitis B virus (HBV)-infected cells, provides a perfect target for therapeutic drugs. In order to mediate successful targeted delivery of these therapies, it is essential to have antibodies that recognize HBsAg with high specificity and affinity. In this report, we constructed a natural immune antigen binding fragments (Fab) antibody phage display library against HBsAg and after three rounds of panning, five Fab fragments with significant HBsAg binding ability were selected and analysed. DNA sequencing revealed that all the light chains had the same sequence, while all the Fd genes exhibited different sequences. For further application, all of the Fab antibodies were reconstructed into single chain antibodies (scFvs) and expressed in Escherichia coli BL21 cells. Indirect enzyme-linked immunosorbent assay analysis demonstrated that all five scFvs maintained a high affinity for HBsAg and could bind HBsAg on the membrane of HBV-infected cells. Indirect fluorescent staining analysis revealed that one of the scFvs (scFv15) could be internalized into HBsAg-positive HepG2.2.15 cells through clathrin-mediated endocytosis pathway. The internalizing scFv15 antibody would have great potential for the targeted delivery of therapeutics to HBV-infected cells.

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“…It has been shown that when the diversity of an antibody phage display library reaches 1×10 7 individual clones, it will contain 99% of all antibody molecules. The size of antibody libraries that have been constructed so far generally ranges between 10 6 and 10 8 clones [24,25]. The size of our library for B cell lymphoma is ~1.94×10 7 , which is of medium size and should satisfy the needs for covering the diversity of antibodies [26].…”

Section: Discussionmentioning

confidence: 99%

Generation and selection of immunized Fab phage display library against human B cell lymphoma

et al. 2007

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npgThe approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×10 7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (V H ) and variable light domains (V L ) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

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Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

Abstract: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

Cited by 11 publications

References 33 publications

Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display

Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display

An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells

Generation and selection of immunized Fab phage display library against human B cell lymphoma

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