Antibody Engineering Volume 2 (Springer Protocols)

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This detailed new edition provides complete and easy access to a variety of antibody engineering techniques. The volume explores topics such as the generation of native, synthetic, or immune antibody libraries, the selection of lead candidates via the different powerful and innovative display technologies, Fc engineering, as well as their production, characterization, and optimization of antibodies.

Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Antibody Engineering: Methods and Protocols, Third Edition presents the reader with an extensive toolbox to create the powerful molecules of tomorrow.

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Date of Birth. Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Month January February March April May June July August September October November December Year Please fill in a complete birthday Enter a valid birthday. The combination of these methods is suitable for routine use in any laboratory familiar with hybridoma cell lines, and can be used to eliminate the risk of losing the genetic information of unique antibodies.

Antibody Engineering Volume 2 Springer Protocols

V-region rescue and sequence analysis is helpful to avoid working with apparently different clones producing identical mAbs as a result of clone proliferation during the limiting dilution steps used in the hybridoma generation [ 33 ]. Amplifying the V regions of antibodies starting off with mRNA isolation from hybridoma cells can be a labour-intensive process, but the here-used method reduces the workload to a minimum while ensuring that V-region sequences are rescued with high efficiency and accuracy, because as reported by Tiller et al.

Therefore, it can be expected that the rescue efficiency based on cDNA from a larger amount of cells will be significantly higher. Sequence mutations have to be avoided, because even one single substitution within antibody FWRs may reduce or even completely abolish antigen binding [ 34 ]. De Haard et al. It is, therefore, important to verify that the rescued sequence truly represents genetic information that determines the affinity of the corresponding antibody.

An alternative approach to confirm the rescued V H -sequence is the use of degenerate primers in combination with N-terminal peptide sequencing of the original murine antibody [ 35 ]. This method is feasible, but more expensive and often not directly available. All of these have assembled comprehensive antibody germ-line sequence data. V H -FWR1 residues could be verified to match the corresponding germ-line sequence, which generally provides the best guess regarding the respective residue. Although that at least in the human system V H -FWR1 mutations can occur in heavily affinity matured antibodies even at the highly conserved position 6, the V H -FWR1 region is the region which seems to be moderately affected by somatic hypermutation when compared to other regions [ 38 , 39 ].

Furthermore, the rescued sequences can be used to generate any recombinant antibody format, including scFv, Fab, F ab 2 and full-size antibodies featuring Fc-regions of the required species and isotype. Even though these formats can be generated more quickly if the confirmation of the V-region rescue by the here-suggested analyses is omitted, but the results may be less satisfactory, since uncorrected primer-derived mutations may lead to loss of epitope-specificity and affinity as well as problems with expression yields and solubility [ 12 ].

The analysis of recombinant mAbs based on the rescued V regions circumvents these drawbacks by allowing the direct comparison of the antigen-binding properties of the rescued antibody and its original hybridoma-produced counterpart. Additionally, the recombinant format of the antibody allows a completely new therapeutic approach in the malaria field, anti-malarial antibody fusion proteins.

Antibody Engineering Volume 2

In such an antibody fusion protein, an scFv is genetically fused to a protein with anti-malarial activity. The resulting protein has a significantly increased activity and therefore meets the criteria for a potential therapeutic agent [ 40 ].


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The here-applied plant-based transient expression of a mouse-human chimeric full-size antibody has been shown to yield a functional mAb at reduced costs and saved time [ 41 ]. In mammalian expression systems, very high yields are only achieved after cost- and time-consuming cell line development and carefully regulated cultivation of cells [ 42 ]. Furthermore, the agro-infiltration-based transient plant expression system provides the opportunity to easily express different recombinant proteins e.

Antibody Engineering - Methods and Protocols | Damien Nevoltris | Springer

Different leaves of the same plant can be used to efficiently compare independent mAbs as well as different combinations of antibody heavy and light chains or mutants thereof, making this system ideally suited for the time-efficient production and analysis of antibody sequences derived from a hybridoma antibody cloning approach. In the context of malaria research, antibodies can be used for the functional analysis of potential vaccine targets, as well as therapeutic molecules. Besides the determination of epitopes and antigen-binding characteristics, the analysis of parasite growth inhibitory efficacy in various in vitro assays, including the blood-stage growth inhibition assay GIA , transmission blocking assays TBA or the inhibition of sporozoite invasion ISI , is essential to asses the suitability of a mAb for the desired application.

Therefore it is beneficial if the mAbs can be efficiently produced at mg quantities at low cost in one single batch to facilitate the concurrent screening of several mAbs with potential for the desired downstream application. If assays including cellular immune responses are the objective of further investigations besides blocking activity of the mAb, as it is the case in GIAs, TBAs and ISIs, several aspects still need to be considered.


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It has been shown that murine mAbs were successful in protecting mice against parasite-challenge, but their corresponding chimeric mAbs bearing human Fc regions were not [ 43 ]. This indicates the importance of the IgG-Fc region for protection in its parental species. For human applications, fully human mAbs have the significant advantage of not inducing any human anti-mouse immune responses, like fully murine, and also mouse-human chimeric mAbs do, as introduced in the background section.

Therefore, a homologous workflow for the generation of fully human antibodies is currently being set up at the Fraunhofer-Institute for Molecular Biology and Applied Ecology IME Aachen, Germany [ 45 ]. If these applications are finally striven for, either a mammalian expression system may be chosen, or genetically modified plants which attach a humanized IgG-Fc N-glycan profile. It has recently been discussed, whether plant-derived mAbs are applicable acute-phase drugs, especially against the viral disease Ebola, one of the major acute burdens in Western Africa [ 48 , 49 ].

Indisputable, there is still many research to be done to meet the criteria for a cost-efficient production of a final, especially mAb-based, therapeutic against poverty-related diseases, such as ebola, or malaria. Antibody V-region rescue is an essential technique to preserve the unique epitope binding specificity of mAbs.

The complete workflow is described in detail, including initial V-region amplification, cloning, heterologous expression in Nicotiana benthamiana and verification of the rescued sequences by comparative functional analyses. The described method can be applied to facilitate the rescue, production and characterization of larger numbers of monoclonal antibodies.

This is especially relevant in the field of malaria research where the plurality of P. Trial watch: monoclonal antibodies in cancer therapy. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.

Instability of a hybridoma cell line in a homogeneous continuous perfusion culture system. Cloning of variable domains from mouse hybridoma by PCR. Antibody engineering. Berlin: Springer; Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. Opinion: antibody-based therapies for malaria. Nat Rev Microbiol. Ducancel F, Muller BH. Molecular engineering of antibodies for therapeutic and diagnostic purposes. Cloning and expression of murine Ig genes from single B cells. J Immunol Methods. Breitling F, Dubel S. Cloning and expression of single-chain fragments scfv from mouse and rat hybridomas.

Methods Mol Med. Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction.

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Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity. Protein Eng.


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Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Infect Immun. Malaria vaccine candidate antigen targeting the pre erythrocytic stage of Plasmodium falciparum produced at high-level in plants. Biotechnol J.