Affinity Maturation of Monoclonal Antibody 1E11 by Targeted Randomization in CDR3 Regions Optimizes Therapeutic Antibody Targeting of HER2-Positive Gastric Cancer

Ko, Bong-Kook; Choi, Soyoung; Cui, Lei; Lee, Young Ha; Hwang, In Sik; Kim, Kyu Tae; Shim, Hyunbo; Lee, Jong Seo

doi:10.1371/journal.pone.0134600

Cited by 8 publications

(11 citation statements)

References 39 publications

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“…Thus we may conclude that for this set of Abs, the CDR-Hs largely drive binding. However, without such experimental data, it is not obvious how important each CDR is to the binding profile; e.g., there are certainly cases where light chains are very important for strong binding ( Ko et al, 2015 ). Unfortunately, simply comparing overall CDR sequence similarity (‘all’ in Figure 5—figure supplement 3 ), as might be appropriate without any assumptions, yields a pattern that doesn’t reflect binning as well as that for individual CDR-Hs.…”

Section: Resultsmentioning

confidence: 99%

Computationally-driven identification of antibody epitopes

Hua

Gacerez

Sentman

et al. 2017

eLife

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Understanding where antibodies recognize antigens can help define mechanisms of action and provide insights into progression of immune responses. We investigate the extent to which information about binding specificity implicitly encoded in amino acid sequence can be leveraged to identify antibody epitopes. In computationally-driven epitope localization, possible antibody–antigen binding modes are modeled, and targeted panels of antigen variants are designed to experimentally test these hypotheses. Prospective application of this approach to two antibodies enabled epitope localization using five or fewer variants per antibody, or alternatively, a six-variant panel for both simultaneously. Retrospective analysis of a variety of antibodies and antigens demonstrated an almost 90% success rate with an average of three antigen variants, further supporting the observation that the combination of computational modeling and protein design can reveal key determinants of antibody–antigen binding and enable efficient studies of collections of antibodies identified from polyclonal samples or engineered libraries.

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Section: Resultsmentioning

confidence: 99%

Computationally-driven identification of antibody epitopes

Hua

Gacerez

Sentman

et al. 2017

eLife

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“…e affinity maturation technologies mimic the in vivo antibody maturation occurring in B cells during the immune response [9][10][11], but achieving higher affinities (from 10 − 10 to 10 − 15 mol/L) than those obtained in vivo (about 10 − 10 mol/ L) [12][13][14]. ese technologies are based on random mutation of the antibody binding sites [15][16][17][18][19][20] and the subsequent selection of the antibody variants showing the highest affinity for the target, using a variety of display methods (yeast surface display, phage display, E. coli surface display, mammalian cell display, ribosome display and mRNA display) [21][22][23]. Among them, yeast surface display (YSD) is the most widely used affinity maturation platform because it combines a lot of advantages compared with the other approaches [24][25][26][27]; in particular, the eukaryotic machinery ensures the correct folding and post-translational modifications of the displayed proteins; in addition, the in vivo yeast recombination is more efficient than in vitro cloning by ligation for the library generation [28]; more importantly, above all, clones with improved affinity can be selected by fluorescence-activated cell sorting (FACS).…”

Section: Introductionmentioning

confidence: 99%

Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

Cembrola

Ruzza

Troise

et al. 2019

BioMed Research International

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The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

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“…2a). The targeted residues were randomized using hand-mixed spiked oligonucleotides, which were designed to randomly mutate each residue while retaining the parental amino acids at a level of ~50% at each residue 25,26 , to conserve parental residues critically contributing to IL-4Rα binding (Fig. 2a).…”

Section: Resultsmentioning

confidence: 99%

“…Affinity maturation of Abs by CDR mutagenesis was conducted in the scFab format containing a linker with 63 amino acids between the light chain and the heavy chain 27 . In the affinity maturation of 4R34, the targeted residues in VH-CDR3 (residues 95–98), VH-CDR2 (residues 50, 52–57), and VL-CDR3 (residues 89, 90, 93, 95a) were randomized using hand-mixed spiked oligonucleotides 25,26 , as described in the text. In the affinity maturation of 4R34.1, the targeted residues of VL-CDR1 (residues 27–32) and VH-CDR1 (residues 31–35) were randomized with a degenerate codon NNK 26 .…”

Section: Methodsmentioning

confidence: 99%

Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

Kim

Jung

Kim

et al. 2019

Sci Rep

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Development of antagonistic antibody (Ab) against interleukin-4 receptor alpha (IL-4Rα) subunit of IL-4/IL-13 receptors is a promising therapeutic strategy for T helper 2 (T H 2)-mediated allergic diseases such as asthma and atopic dermatitis. Here we isolated anti-human IL-4Rα antagonistic Abs from a large yeast surface-displayed human Ab library and further engineered their complementarity-determining regions to improve the affinity using yeast display technology, finally generating a candidate Ab, 4R34.1.19. When reformatted as human IgG1 form, 4R34.1.19 specifically bound to IL-4Rα with a high affinity ( K D ≈ 178 pM) and effectively blocked IL-4- and IL-13-dependent signaling in a reporter cell system at a comparable level to that of the clinically approved anti-IL-4Rα dupilumab Ab analogue. Epitope mapping by alanine scanning mutagenesis revealed that 4R34.1.19 mainly bound to IL-4 binding sites on IL-4Rα with different epitopes from those of dupilumab analogue. Further, 4R34.1.19 efficiently inhibited IL-4-dependent proliferation of T cells among human peripheral blood mononuclear cells and suppressed the differentiation of naïve CD4 + T cells from healthy donors and asthmatic patients into T H 2 cells, the activities of which were comparable to those of dupilumab analogue. Our work demonstrates that both affinity and epitope are critical factors for the efficacy of anti-IL-4Rα antagonistic Abs.

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Affinity Maturation of Monoclonal Antibody 1E11 by Targeted Randomization in CDR3 Regions Optimizes Therapeutic Antibody Targeting of HER2-Positive Gastric Cancer

Cited by 8 publications

References 39 publications

Computationally-driven identification of antibody epitopes

Computationally-driven identification of antibody epitopes

Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

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