Affinity maturation of antibodies assisted by
            <i>in silico</i>
            modeling

Barderas, Rodrigo; Desmet, Jan; Timmerman, Peter; Meloen, Rob H.; Casal, J. Ignacio

doi:10.1073/pnas.0801221105

Cited by 125 publications

(70 citation statements)

References 39 publications

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“…This finding correlates with the observation made in previous studies that antigen-antibody interfaces are more planar and significantly well-ordered or packed (37). Methods that adopt CDR loop randomization strategies for affinity maturation (38) typically focus on heavy chain, especially CDR H3, because this loop accounts for most of the stabilizing contacts in many cases. Our results with 4E11 show that diversification strategies may benefit from a rational approach and that incorporating VL-loops for targeted diversification may further aid affinity maturation.…”

Section: Discussionsupporting

confidence: 79%

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

Tharakaraman¹,

Robinson²,

Hatas³

et al. 2013

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

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Affinity improvement of proteins, including antibodies, by computational chemistry broadly relies on physics-based energy functions coupled with refinement. However, achieving significant enhancement of binding affinity (>10-fold) remains a challenging exercise, particularly for cross-reactive antibodies. We describe here an empirical approach that captures key physicochemical features common to antigen-antibody interfaces to predict proteinprotein interaction and mutations that confer increased affinity. We apply this approach to the design of affinity-enhancing mutations in 4E11, a potent cross-reactive neutralizing antibody to dengue virus (DV), without a crystal structure. Combination of predicted mutations led to a 450-fold improvement in affinity to serotype 4 of DV while preserving, or modestly increasing, affinity to serotypes 1-3 of DV. We show that increased affinity resulted in strong in vitro neutralizing activity to all four serotypes, and that the redesigned antibody has potent antiviral activity in a mouse model of DV challenge. Our findings demonstrate an empirical computational chemistry approach for improving protein-protein docking and engineering antibody affinity, which will help accelerate the development of clinically relevant antibodies. (1). Engineering improved affinity and specificity of these compounds can augment their potency and safety while decreasing required dosages. Production of antibodies with binding properties of interest typically relies on methods involving screening large numbers of clones generated by the immune system or by mutant libraries (2, 3). Alternatively, computer-based design offers the potential to rationally mutate available antibodies for improved properties, including enhanced affinity and specificity to target antigens. Recently, several successful examples of antibody affinity improvement by computational methods using physical modeling with energy minimization have been described (4-6). However, such approaches require a 3D structure of the antibody-antigen complex and rarely result in affinity gains greater than 10-fold. Further, these approaches are sensitive to precise atomic coordinates, rendering them inapplicable to computer-generated models. More significantly, enhancement of affinity in the context of an antibody that recognizes multiple antigens (i.e., crossreactive) remains a particular challenge.Dengue is the most medically relevant arboviral disease in humans, with an estimated 3.6 billion people at risk for infection. More than 200 million infections of dengue virus (DV) are estimated to occur globally each year (7). The incidence, geographical outreach, and number of severe disease cases of dengue are increasing (8, 9), making DV of increasing concern as a human pathogen. The complex of DVs is composed of four distinct serotypes (designated DV1-4) (10), which vary from one another at the amino acid level by 25-40%. The sequence and antigenic variability of DVs have challenged efforts to develop an effective vaccine or therapeutic against a...

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Section: Discussionsupporting

confidence: 79%

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

Tharakaraman¹,

Robinson²,

Hatas³

et al. 2013

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

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“…After clarifying by centrifugation at 12,000 ϫ g for 15 min, protein concentrations were determined with the 2-D Quant kit (GE Healthcare). For Western blot, 50 g of protein extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Hybond-C Extra) according to standard procedures (37). After blocking, membranes were incubated overnight at 4°C with PIM1 (dilution, 1:100), MAPKAPK3 (dilution, 1: 500), and ACVR2B (dilution, 1:200) antibodies.…”

Section: Methodsmentioning

confidence: 99%

Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

Babel

Barderas

Dı́az-Uriarte

et al. 2009

Molecular & Cellular Proteomics

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146

143

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There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Fortythree proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers. Molecular & Cellular Proteomics 8:2382-2395, 2009.

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“…This is not surprising, since antibodies selected in pannings by phage display technology normally do not demonstrate very high binding affinity. To increase the binders' affinity, the subsequent step of affinity maturation is required (36)(37). However, this was not a goal of the present study.…”

Section: Resultsmentioning

confidence: 59%

Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

Stuknytė

Brockmann

Huovinen

et al. 2014

Appl Environ Microbiol

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bSingle-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L. helveticus MIMLh5 and one was also capable of binding to the S-layer protein of L. helveticus ATCC 15009. All five anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the Slayer protein of L. helveticus MIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.

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Affinity maturation of antibodies assisted by in silico modeling

Cited by 125 publications

References 39 publications

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

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