Functional Characterization of Two scFv-Fc Antibodies from an HIV Controller Selected on Soluble HIV-1 Env Complexes: A Neutralizing V3- and a Trimer-Specific gp41 Antibody

Trott, Maria; Weiß, Svenja; Antoni, Sascha; Koch, Joachim; Briesen, Hagen von; Hust, Michael; Dietrich, Ursula

doi:10.1371/journal.pone.0097478

Cited by 32 publications

(15 citation statements)

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“…Immune libraries from patients are suited to select specific antibodies against a disease or pathogen, e.g. cancer [ 26 , 27 ], human immunodeficiency virus [ 28 ]or herpes simplex virus [ 29 ]. “Single-pot” libraries allow the selection of antibodies - in theory - against any target.…”

Section: Introductionmentioning

confidence: 99%

Generation and analysis of the improved human HAL9/10 antibody phage display libraries

Wilke²,

et al. 2015

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BackgroundAntibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library.ResultsThe naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations.ConclusionThe highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

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Section: Introductionmentioning

confidence: 99%

Generation and analysis of the improved human HAL9/10 antibody phage display libraries

Wilke²,

et al. 2015

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“…70 µg env DNA and 35 µg furin DNA (Imagenes-bio, Germany) were used to transiently transfect 2 × 10 7 293T cells (ATCC® CRL-11268™) using 210 µl polyethylenimine (PEI, 1 mg/ml, Sigma-Aldrich). Supernatants were harvested after 48 hours and gp140 SOSIP proteins were purified via lectin ( Galanthus nivalis , Sigma-Aldrich) affinity chromatography (elution with 10 ml 0.5 M methyl-α-D-mannopyranoside) 63 . Trimeric gp140 was further purified by size exclusion (HiLoad™ 16/60 Superdex™ 200, GE Healthcare) and anion exchange chromatography (HiTrap™ DEAE FF, GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers

Koch

Kalusche

Torres

et al. 2017

Sci Rep

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Broadly neutralizing antibodies (bnAbs) against HIV-1 protect from infection and reduce viral load upon therapeutic applications. However no vaccine was able so far to induce bnAbs demanding their expensive biotechnological production. For clinical applications, nanobodies (VHH) derived from heavy chain only antibodies from Camelidae, may be better suited due to their small size, high solubility/stability and extensive homology to human VH3 genes. Here we selected broadly neutralizing nanobodies by phage display after immunization of dromedaries with different soluble trimeric envelope proteins derived from HIV-1 subtype C. We identified 25 distinct VHH families binding trimeric Env, of which 6 neutralized heterologous primary isolates of various HIV-1 subtypes in a standardized in vitro neutralization assay. The complementary neutralization pattern of two selected VHHs in combination covers 19 out of 21 HIV-1 strains from a standardized panel of epidemiologically relevant HIV-1 subtypes. The CD4 binding site was preferentially targeted by the broadly neutralizing VHHs as determined by competition ELISAs and 3D models of VHH-Env complexes derived from negative stain electron microscopy. The nanobodies identified here are excellent candidates for further preclinical/clinical development for prophylactic and therapeutic applications due to their potency and their complementary neutralization patterns covering the majority of epidemiologically relevant HIV-1 subtypes.

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“…There are quite different philosophies and strategies for their generation. The easiest approach is the generation of 'immune libraries' from blood samples of immunized human donors [31,30]. Of course this strategy is particularly useful to obtain antibodies against the antigen against which the donors have already developed a specific B-cell reaction.…”

Section: Phage Display Librariesmentioning

confidence: 99%

Designing Human Antibodies by Phage Display

Frenzel

Kügler²,

Helmsing

et al. 2017

Transfus Med Hemother

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ever, are recognized as foreign proteins by humans and therefore induce immune reactions against the therapeutic reagent (human anti-mouse antibody; HAMA) [3]. A solution to this problem was only brought by recombinant DNA technology. Using genetic engineering, the murine sequences were exchanged in parts of the molecule by the homologous human sequences. The result were 'chimeric' (with murine constant domains replaced by human constant domains) or 'humanized' antibodies (only the antigen-binding loops / complementarity determining regions (CDRs) were transferred to human variable region frameworks) [4,5]. These engineered mouse antibodies dominated the first decade of therapeutic antibody approvals, while today it is preferred to use completely human antibodies from the start, thanks to a number of novel technologies allowing to identify them without the necessity to immunize humans. These are on the one hand transgenic animals, typically mice or rats, which carry genetically introduced human immunoglobulin loci while their own antibody genes were knocked out [6][7][8]. Consequently, after immunization they utilize this sequence repertoire to generate IgG from human germ-line sequences, thereby allowing to generate monoclonals by means of classical hybridoma technology. An even more radical way was introduced by the development of antibody phage display, which allowed for the first time to generate human antibodies completely in the test tube and without immunization. Inspired by the work of G.P. Smith on peptide display using filamentous phage [9], antibody phage display was development independently in Heidelberg by Breitling and Dübel [10], in Cambridge by McCafferty et al. [11], and in California by Barbas et al. [12]. The method is based on a physical link between function (antigen binding) and information (antibody gene) in a nanoparticle (the phage virus particle). To achieve this, the antigen binding parts of the antibodies, either as Fab (fragment antigen binding) [13,14] or scFv (single chain fragment variable) [15][16][17], are genetically linked to the surface protein III (pIII) of the M13 phage and thus expressed on the surface of the virus particle. Mixtures of such phage particles, each enKeywords Human monoclonal antibodies · Phage display · Immunotherapy · In vitro evolution · Panning · scFv Summary With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by phage display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategie...

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Functional Characterization of Two scFv-Fc Antibodies from an HIV Controller Selected on Soluble HIV-1 Env Complexes: A Neutralizing V3- and a Trimer-Specific gp41 Antibody

Cited by 32 publications

References 87 publications

Generation and analysis of the improved human HAL9/10 antibody phage display libraries

Generation and analysis of the improved human HAL9/10 antibody phage display libraries

Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers

Designing Human Antibodies by Phage Display

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