scholarly journals A Two-Step Approach for the Design and Generation of Nanobodies

2018 ◽  
Vol 19 (11) ◽  
pp. 3444 ◽  
Author(s):  
Hanna Wagner ◽  
Sarah Wehrle ◽  
Etienne Weiss ◽  
Marco Cavallari ◽  
Wilfried Weber
Keyword(s):  
Heavy Chain ◽  
Animal Experiments ◽  
Affinity Maturation ◽  
Phage Library ◽  
Second Step ◽  
Selection Procedures ◽  
Variable Domains ◽  
Phage Libraries ◽  
Complementarity Determining Regions ◽  
Conventional Antibody

Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.

The diversity of rearranged immunoglobulin heavy chain variable region genes in peripheral blood B cells of preterm infants is restricted by short third complementarity-determining regions but not by limited gene segment usage

Blood ◽  
2001 ◽  
Vol 97 (5) ◽  
pp. 1511-1513 ◽  
Author(s):  
Michael Zemlin ◽  
Karl Bauer ◽  
Michael Hummel ◽  
Sabine Pfeiffer ◽  
Simone Devers ◽  
...  
Keyword(s):  
B Cells ◽  
Preterm Infants ◽  
Heavy Chain ◽  
Peripheral Blood ◽  
Gene Segment ◽  
Immunoglobulin Heavy Chain ◽  
Term Infants ◽  
Extremely Preterm ◽  
Extremely Preterm Infants ◽  
Complementarity Determining Regions

The immunoglobulin diversity is restricted in fetal liver B cells. This study examined whether peripheral blood B cells of extremely preterm infants show similar restrictions (overrepresentation of some gene segments, short third complementarity-determining regions [CDR3]). DNA of rearranged immunoglobulin heavy chain genes was amplified by polymerase chain reaction, cloned, and sequenced. A total of 417 sequences were analyzed from 6 preterm infants (25-28 weeks of gestation), 6 term infants, and 6 adults. Gene segments from the entire VHand DH gene locus were rearranged in preterm infants, even though the DH7-27 segment was overrepresented (17% of rearrangements) compared to term infants (7%) and adults (2%). CDR3 was shorter in preterm infants (40 ± 10 nucleotides) than in term infants (44 ± 12) and adults (48 ± 14) (P < .001) due to shorter N regions. Somatic mutations were exclusively found in term neonates and adults (mutational frequency 0.8% and 1.8%). We conclude that preterm infants have no limitations in gene segment usage, whereas the diversity of CDR3 is restricted throughout gestation.

scholarly journals Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains

2014 ◽  
Vol 111 (28) ◽  
pp. 10275-10280 ◽  
Author(s):  
D. Fera ◽  
A. G. Schmidt ◽  
B. F. Haynes ◽  
F. Gao ◽  
H.-X. Liao ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lineage ◽  
Affinity Maturation ◽  
Variable Domains

scholarly journals Generation of antibody-based therapeutics targeting the idiotype of B-cell malignancies

2018 ◽  
Vol 2 (1) ◽  
pp. 12-21
Author(s):  
Emily Weiss ◽  
Robert Sarnovsky ◽  
Mitchell Ho ◽  
Evgeny Arons ◽  
Robert Kreitman ◽  
...  
Keyword(s):  
B Cell ◽  
Specific Binding ◽  
Phage Library ◽  
Selection Strategy ◽  
Proof Of Concept ◽  
Single Chain ◽  
Human Igg1 ◽  
Specificity Of Binding ◽  
Complementarity Determining Regions ◽  
Phage Selection

ABSTRACT Background A feature of many B-cell tumors is a surface-expressed immunoglobulin (sIg). The complementarity-determining regions (CDRs) of the sIg, termed the ‘idiotype’, are unique to each tumor. We report on a phage selection strategy to generate anti-idiotype therapeutics that reacts with sIg CDR3 sequences; the MEC1 B-cell tumor line was used as proof of concept. Methods To create a mimetic of the MEC1 idiotype, CDR3 sequences from heavy and light chains of the sIg were grafted into a single chain variable fragment (scFv) framework scaffold. Using the Tomlinson I phage library of human scFvs, we enriched for binders to MEC1 CDR3 sequences over unrelated CDR3 sequences. Results By ELISA we identified 10 binder phages. Of these, five were sequenced, found to be unique and characterized further. By flow cytometry each of the five phages bound to MEC1 cells, albeit with different patterns of reactivity. To establish specificity of binding and utility, the scFv sequences from two of these binders (phages 1 and 7) were converted into antibody-toxin fusion proteins (immunotoxins) and also cloned into a human IgG1 expression vector. Binders 1 and 7 immunotoxins exhibited specific killing of MEC1 cells with little toxicity for non-target B-cell lines. The full-length antibody recreated from the binder-1 scFv also exhibited specific binding. Conclusion Our results establish the utility of using engrafted CDR3 sequences for selecting phage that recognize the idiotype of B-cell tumors.

scholarly journals Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation

2020 ◽  
Vol 117 (29) ◽  
pp. 16949-16960 ◽  
Author(s):  
Denice T. Y. Chan ◽  
Lesley Jenkinson ◽  
Stuart W. Haynes ◽  
Mark Austin ◽  
Agata Diamandakis ◽  
...  
Keyword(s):  
Structural Changes ◽  
Structural Evolution ◽  
Affinity Maturation ◽  
Antibody Engineering ◽  
Inhibitory Antibody ◽  
Extensive Sequence ◽  
Shape Complementarity ◽  
Complementarity Determining Regions ◽  
Inhibition Potency

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

Effects of a long-term spaceflight on immunoglobulin heavy chains of the urodele amphibian Pleurodeles waltl

2005 ◽  
Vol 98 (3) ◽  
pp. 905-910 ◽  
Author(s):  
Rachel Boxio ◽  
Christian Dournon ◽  
Jean-Pol Frippiat
Keyword(s):  
Heavy Chain ◽  
Oral Immunization ◽  
Northern Blotting ◽  
Ground Control ◽  
Heavy Chains ◽  
Immune Parameters ◽  
Variable Domains ◽  
Pleurodeles Waltl ◽  
Antibody Heavy Chain

A variety of immune parameters are modified during and after a spaceflight. The effects of spaceflights on cellular immunity are well documented; however, little is known about the effects of these flights on humoral immunity. During the Genesis space experiment, two adult Pleurodeles waltl (urodele amphibian) stayed 5 mo onboard Mir and were subjected to oral immunization. Animals were killed 10 days after their return to earth. IgM and IgY heavy-chain transcripts in their spleens were quantified by Northern blotting. The use of the different VH families (coding for antibody heavy-chain variable domains) in IgM heavy chain transcripts was also analyzed. Results were compared with those obtained with ground control animals and animals reared in classical conditions in our animal facilities. We observed that, 10 days after the return on earth, the level of IgM heavy-chain transcription was normal but the level of IgY heavy-chain transcription was at least three times higher than in control animals. We also observed that the use of the different VH families in IgM heavy-chain transcripts was modified by the flight. These data suggest that the spaceflight affected the antibody response against the antigens contained in the food.

Ig L-chain Shuffling for Affinity Maturation of Phage Library-derived Human Anti-human MCP-1 Antibody Blocking its Chemotactic Activity

2008 ◽  
Vol 143 (5) ◽  
pp. 593-601 ◽  
Author(s):  
K. Yoshinaga ◽  
M. Matsumoto ◽  
M. Torikai ◽  
K. Sugyo ◽  
S. Kuroki ◽  
...  
Keyword(s):  
Affinity Maturation ◽  
Chemotactic Activity ◽  
Phage Library

Camelid Heavy-Chain Variable Domains Provide Efficient Combining Sites to Haptens†

Biochemistry ◽  
2000 ◽  
Vol 39 (6) ◽  
pp. 1217-1222 ◽  
Author(s):  
Silvia Spinelli ◽  
Leon G. J. Frenken ◽  
Pim Hermans ◽  
Theo Verrips ◽  
Kieron Brown ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Variable Domains

scholarly journals Synthetic peptides corresponding to third hypervariable region of human monoclonal IgM rheumatoid factor heavy chains define an immunodominant idiotype.

1985 ◽  
Vol 162 (2) ◽  
pp. 756-761 ◽  
Author(s):  
R D Goldfien ◽  
P P Chen ◽  
S Fong ◽  
D A Carson
Keyword(s):  
Rheumatoid Factor ◽  
Heavy Chain ◽  
Synthetic Peptides ◽  
Gene Segment ◽  
Hypervariable Region ◽  
Heavy Chains ◽  
The Third ◽  
Specific Induction ◽  
Antiidiotypic Antibodies ◽  
Complementarity Determining Regions

Synthetic peptides corresponding to eight individual heavy chain complementarity-determining regions (CDR) of three human monoclonal IgM anti-IgG (rheumatoid factor [RF]) paraproteins elicited rabbit antibodies with markedly different properties. All antisera recognized the immunizing peptide, and several reacted with the isolated IgM heavy chain on immunoblots. However, only the antisera against peptides representing the third CDR bound consistently and specifically to the intact IgM-RF molecule. These data indicate that the third CDR of human mu chains comprises an immunodominant idiotype, and suggest that the D gene segment may be especially important in creating idiotypic diversity. Synthetic peptides corresponding to the third heavy chain CDR of human paraproteins may be clinically useful for the specific induction of antiidiotypic antibodies.

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