The improvement of an anti-CD22 immunotoxin

Kawa, Seiji; Onda, Masanori; Ho, Mitchell; Kreitman, Robert J.; Bera, Tapan K.; Pastan, Ira

doi:10.4161/mabs.3.5.17228

Cited by 15 publications

(4 citation statements)

References 42 publications

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“…Four of the disruptive mutations (N52 and K56 in CDR2 and Y100 and W100e in CDR3) were identified in our computational alanine scanning mutagenesis (Table S1 in Supplementary Material). These and other previous results ( 39 , 53 , 54 ) highlight the value of alanine scanning mutagenesis to identify permissive CDR sites that can be mutated during antibody affinity maturation.…”

Section: Resultssupporting

confidence: 80%

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

Tiller

Chowdhury

et al. 2017

Front. Immunol.

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The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies.

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Section: Resultssupporting

confidence: 80%

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

Tiller

Chowdhury

et al. 2017

Front. Immunol.

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“…However, the optimization of the physicochemical properties of an antibody without perturbing the affinity and specificity for the cognate molecule is a challenging endeavor. Progress in this field is accelerating, and successful examples of improved antibodies using biobetter strategies have been reported, such as higher affinity for the cognate, increased stability in solution, enhanced pharmacokinetics, diminished immunogenicity, or conjugation to drug delivery systems [5] – [9] . Nevertheless, to advance further the deployment of biobetter strategies in the design and preparation of improved therapeutics it is necessary to strengthen our understanding of the physicochemical properties of engineered antibodies [10] .…”

Section: Introductionmentioning

confidence: 99%

Affinity Improvement of a Therapeutic Antibody by Structure-Based Computational Design: Generation of Electrostatic Interactions in the Transition State Stabilizes the Antibody-Antigen Complex

et al. 2014

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The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

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“…To further investigate the critical residues for antigen-antibody interactions, alanine scanning mutagenesis was carried out in both CDRs of heavy and light chains 34 . Each CDR non-alanine residue in 8D3 was mutated to alanine by site-directed mutagenesis.…”

Section: Resultsmentioning

confidence: 99%

Improvement in affinity and thermostability of a fully human antibody against interleukin-17A by yeast-display technology and CDR grafting

Sun

Yang

Lin

et al. 2019

Acta Pharmaceutica Sinica B

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Monoclonal antibodies (mAbs) are widely used in many fields due to their high specificity and ability to recognize a broad range of antigens. IL-17A can induce a rapid inflammatory response both alone and synergistically with other proinflammatory cytokines. Accumulating evidence suggests that therapeutic intervention of IL-17A signaling offers an attractive treatment option for autoimmune diseases and cancer. Here, we present a combinatorial approach for optimizing the affinity and thermostability of a novel anti-hIL-17A antibody. From a large naïve phage-displayed library, we isolated the anti-IL-17A mAb 7H9 that can neutralize the effects of recombinant human IL-17A. However, the modest neutralization potency and poor thermostability limit its therapeutic applications. In vitro affinity optimization was then used to generate 8D3 by using yeast-displayed random mutagenesis libraries. This resulted in four key amino acid changes and provided an approximately 15-fold potency increase in a cell-based neutralization assay. Complementarity-determining regions (CDRs) of 8D3 were further grafted onto the stable framework of the huFv 4D5 to improve thermostability. The resulting hybrid antibody 9NT/S has superior stabilization and affinities beyond its original antibody. Human fibrosarcoma cell-based assays and in vivo analyses in mice indicated that the anti-IL-17A antibody 9NT/S efficiently inhibited the secretion of IL-17A-induced proinflammatory cytokines. Therefore, this lead anti-IL-17A mAb might be used as a potential best-in-class candidate for treating IL-17A related diseases.

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The improvement of an anti-CD22 immunotoxin

Cited by 15 publications

References 42 publications

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

Affinity Improvement of a Therapeutic Antibody by Structure-Based Computational Design: Generation of Electrostatic Interactions in the Transition State Stabilizes the Antibody-Antigen Complex

Improvement in affinity and thermostability of a fully human antibody against interleukin-17A by yeast-display technology and CDR grafting

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