pFab-CMV, a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies

Sanna, Pietro Paolo; Samson, Maria; Moon, Jenny; Rozenshteyn, Roman; Logu, Alessandro De; Williamson, R. Anthony; Burton, Dennis R.

doi:10.1016/s1380-2933(98)00022-0

Cited by 16 publications

(6 citation statements)

References 12 publications

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“…Furthermore, we have shown that traditional ligation can also be used with our method (Figure 2), hence allowing laboratories to integrate InTag-based IgG reformatting into their current protocols without the need to generate new primer sets. As an example, the InTag cloning strategy can be readily applied to the pFab-CMV vector system (15) by converting the SpeI-XbaI fragment into an InTag adaptor. This can be achieved by inserting an E. coli selection marker (e.g.…”

Section: Discussionmentioning

confidence: 99%

One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification

Chen

Fabri

Wilson

et al. 2013

Nucleic Acids Research

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We describe a novel cloning method, referred to as insert-tagged (InTag) positive selection, for the rapid one-step reformatting of phage-displayed antibody fragments to full-length immunoglobulin Gs (IgGs). InTag positive selection enables recombinant clones of interest to be directly selected without cloning background, bypassing the laborious process of plating out cultures and colony screening and enabling the cloning procedure to be automated and performed in a high-throughput format. This removes a significant bottleneck in the functional screening of phage-derived antibody candidates and enables a large number of clones to be directly reformatted into IgG without the intermediate step of Escherichia coli expression and testing of soluble antibody fragments. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated, and optimized methods for the small-scale transient expression of IgGs at high levels are described. InTag positive selection cloning has the potential for wide application in high-throughput DNA cloning involving multiple inserts, markedly improving the speed and quality of selections from protein libraries.

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Section: Discussionmentioning

confidence: 99%

One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification

Chen

Fabri

Wilson

et al. 2013

Nucleic Acids Research

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“…This recombinant Fab antibody can also be expressed as soluble form using the same expression system. This expression vector is also compatible with the mammalian expression vector pFab-CMV and the light chain and Fd chain can be subcloned from pCOMB3H into pFab-CMV which carries human Fc region for expression of full-length antibody without any modification [50].…”

Section: Discussionmentioning

confidence: 99%

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

et al. 2009

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5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.

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“…In the present study these two chimaeric fragments were further cloned into the mammalian expression vector pFab‐CMV [24]. The essential features of this vector and our cloning strategy are shown in Scheme 1.…”

Section: Methodsmentioning

confidence: 99%

Generation and characterization of a high‐affinity chimaeric antibody against hepatitis B surface antigen

Bose¹,

Khanna²,

Acharya³

et al. 2006

Biotech and App Biochem

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Antibody against HBsAg (hepatitis B surface antigen) is advocated for the passive immunotherapy in certain cases of hepatitis B infections. A recombinant monoclonal antibody against HBsAg would offer several advantages over the currently used polyclonal human hepatitis B immunoglobulin. 5S is a mouse monoclonal antibody that binds to HBsAg with very high affinity. However, this mouse antibody cannot be used for therapeutic purposes, as it may elicit antimouse immune responses. Chimaerization, by replacing mouse constant domains with human counterparts, can reduce the immunogenicity of this molecule. We have cloned the V(H) (heavy-chain variable region) and V(L) (light-chain variable region) genes of this mouse antibody, and fused them with C(H)1 (heavy-chain constant domain 1) of human IgG1 and C(L) (light-chain constant domain) of human kappa chain respectively. These chimaeric genes were cloned into a mammalian expression vector (pFab-CMV), which has a modular cassette coding for part of the hinge, C(H)2 and C(H)3 of human IgG1. The recombinant construct was transfected in CHO (Chinese-hamster ovary) cells to generate a stable transfectoma. The resulting transfectoma was maintained in a serum-free medium and the full-length chimaeric anti-HBsAg antibody was purified from the culture supernatant. The yield of the purified chimaeric antibody was moderate ( approximately 5.5 mg/l). We further characterized the chimaeric antibody using several in vitro techniques. It was observed that the chimaeric molecule was glycosylated and expressed in the expected heterodimeric form. This chimaeric antibody has very high affinity and specificity, similar to that of the original mouse monoclonal antibody.

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pFab-CMV, a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies

Cited by 16 publications

References 12 publications

One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification

One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

Generation and characterization of a high‐affinity chimaeric antibody against hepatitis B surface antigen

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