Synthetic Antibodies Designed on Natural Sequence Landscapes

Zhai, Wenwu; Glanville, Jacob; Fuhrmann, Markus; Mei, Li; Ni, Irene; Sundar, Purnima; Blarcom, Thomas Van; Abdiche, Yasmina; Lindquist, Kevin C.; Strohner, Ralf; Telman, Dilduz; Cappuccilli, Guido; Finlay, William J. J.; Brülle, Jan; Cox, David; Pons, Jaume; Rajpal, Arvind

doi:10.1016/j.jmb.2011.07.018

Cited by 82 publications

(89 citation statements)

References 40 publications

(78 reference statements)

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“…The authors attributed these framework enrichments to increased expression and optimal folding within the periplasm of the Escherichia coli host cells. These findings were further corroborated by the work of Glanville and colleagues (8). We therefore housed our CDR libraries within an scFv framework composed of V H 1-69 and V L 1-44.…”

supporting

confidence: 77%

Construction of a rationally designed antibody platform for sequencing-assisted selection

Larman

Pavlova

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Antibody discovery platforms have become an important source of both therapeutic biomolecules and research reagents. Massively parallel DNA sequencing can be used to assist antibody selection by comprehensively monitoring libraries during selection, thus greatly expanding the power of these systems. We have therefore constructed a rationally designed, fully defined single-chain variable fragment (scFv) library and analysis platform optimized for analysis with short-read deep sequencing. Sequence-defined oligonucleotide libraries encoding three complementarity-determining regions (L3 from the light chain, H2 and H3 from the heavy chain) were synthesized on a programmable microarray and combinatorially cloned into a single scFv framework for molecular display. Our unique complementarity-determining region sequence design optimizes for protein binding by utilizing a hidden Markov model that was trained on all antibody-antigen cocrystal structures in the Protein Data Bank. The resultant ∼10 12-member library was produced in ribosome-display format, and comprehensively analyzed over four rounds of antigen selections by multiplex paired-end Illumina sequencing. The hidden Markov model scFv library generated multiple binders against an emerging cancer antigen and is the basis for a next-generation antibody production platform.antibody display | synthetic antibody library | single framework antibody library A ntibodies are useful for their ability to bind molecular surfaces with high affinity and specificity. The genetic basis for their structural diversity is partially encoded in the germ line, but is also the result of stochastic genetic events, including chromosomal rearrangements, nontemplated nucleotide insertions, and somatic hypermutation. The majority of this diversity is localized to the complementarity-determining regions (CDRs), which are the six-peptide loops that protrude from the variable domain framework to form the antigen-combining surface of the antibody molecule. Three CDR loops are contributed by the heavy chain (H1, H2, and H3) and three by the light chain (L1, L2, and L3). CDRs 1 and 2 are encoded in the germ line, and are thus more constrained in their diversity. L3 is characterized by "junctional diversity," formed during the recombination of two gene segments (V and J). Finally, H3 is formed by two consecutive genetic rearrangements (first between D and J, and then between V and DJ), and is additionally accompanied by nontemplated "N" nucleotides, making this CDR the source of most naturally occurring antibody diversity.Our goal was to develop a synthetic antibody production platform inspired by nature, which could be seamlessly integrated with massively parallel, short-read DNA sequencing analysis (Fig. 1A) (1, 2). For maximum convenience, we required that library amplification and sequencing reactions should depend upon a single set of primers, rather than the complex mixture necessary for natural repertoire amplification and analysis. Like others before, we therefore constructed a highly div...

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supporting

confidence: 77%

Construction of a rationally designed antibody platform for sequencing-assisted selection

Larman

Pavlova

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The parental anti-CXCL13 antibody 3B4 is composed of the V H 1-69 and V 2-14 germlines (13). Domains from the V H 1 and V 2 families have been associated with stable scFv v-domain pairing (31,32), and both V H 1-69 and V 2-14 are highly represented in the human antibody repertoire (33,34). Indeed, an extensive study of V H -V L pairing found V 2-14 to be the most highly represented V germline in the repertoire, where it is often paired with V H 1-69 (34).…”

Section: Discussionmentioning

confidence: 99%

“…5E shows that R93L removes a positively charged patch in the paratope, which may dilute the nearby attractive electrostatic interactions described above and introduce a repulsive interaction with Lys 46 of CXCL13. Leu 93 also makes new intra-domain interactions with both Trp 32 and Tyr 30 in V L -CDR1, leading to increased scFv stability (Fig. 5F).…”

Section: Y91a Is Critical In Driving Affinity Improvements In E10 Scfv-mentioning

confidence: 99%

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Terraube

Tam

et al. 2016

Journal of Biological Chemistry

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Background: Antibody v-domains in scFv format often suffer from aggregation and stability issues that restrict formulation. Results: Structural and empirical analyses of an optimized scFv revealed that three V L -CDR3 mutations were sufficient to mediate significant stability and affinity improvements. Conclusion: scFv issues were resolved via removal of side-chain clashes at the V L /V H interface. Significance: CDR-restricted mutagenesis delivers stability-optimized molecules for high concentration dosing.

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“…Such saturation necessarily encodes all 20 amino acids in ratios dictated by the genetic code. Moreover, this saturation severely limits the functional diversity of such libraries by encoding amino acid composition that is unable to fold natively, and leads to premature termination through the encoding of a stop codon, which in turn can lead to non-functional proteins [5][6][7]. Several solutions to reduce redundancy in the code and exclusion of termination signals has led to inventions such as the 22c trick (all 20 amino acids are encoded by 22 codons through degenerate oligonucleotides), offering near non-degenerate randomization [8].…”

Section: Introductionmentioning

confidence: 99%

“…Analysis of in-vivo antibody repertoires have identified strong positional bias of amino acids tolerated by antibody folding [3,5,6,10]. Additionally, the number, identity and position of residues favored to make antigen contacts in both VH and VL domains varies both according to the type of antigen (protein, peptide or hapten), thus providing information that can be used to design libraries that have a greater proportion of functional members or even to design target-specific antibody libraries [3].…”

Section: Introductionmentioning

confidence: 99%

Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing

Frigotto¹,

Smith²,

Brankin³

et al. 2015

Antibodies

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We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous

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Synthetic Antibodies Designed on Natural Sequence Landscapes

Cited by 82 publications

References 40 publications

Construction of a rationally designed antibody platform for sequencing-assisted selection

Construction of a rationally designed antibody platform for sequencing-assisted selection

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing

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