DOI: 10.5061/dryad.kh18932c8
title: Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles-primary dataset

description:   Interleukin-2 (IL-2) had been been engineered up to now by
 yeast display and in silico rational design. In this article we reshaped
 IL-2 interface with the IL-2 receptor beta subunit to increase binding
 affinity between both interacting partners, using phage display. Multiple
 IL-2 mutated variants were selected from large phage-displayed libraries,
 showing shared molecular patterns. An accumulation of negative charges in
 the segment 81-87 of IL-2 primary sequence was observed, as well as the
 strong preponderance of the replacement I92L. The first feature
 contributed to an optimized electrostatic complementarity between IL-2 and
 IL-2 receptor beta chain, resulting in higher affinity and faster
 association kinetics than the ones of previously reported H9 superkine
 retrieved from yeast display libraries. The presence of a Leu residue at
 position 92 was the key molecular determinant for a favourable biophysical
 profile characterized by high stability and production in mammalian-cell
 based recombinant systems, and decreased aggregation propensity. The new
 beta super-binders behaved as potent super agonists, both in vitro and in
 vivo. The latter scenario showed their better preformance when compared to
 both non-mutated IL-2 and H9. The current dataset contains source data for
 graphics showing frequency mutations and charge distribution among
 unselected variants contained in phage-displayed libraries and selected
 clones enriched after selection on immobilized beta chain. Data showing
 the direct comparison between different IL-2 mutated variants produced as
 Fc-fusion proteins are also presented. The comparison includes the results
 of beta chain binding assays (ELISA), proliferation and phosphorylation
 assays in vitro, in vivo expansion of lymphocyte populations and
 anti-tumor activity in animal models. Taken together, the above described
 data support the unique features of the new beta super-binders and their
 potential as immunostimulatory and anti-cancer agents.
description: The dataset contains primary data used to construct graphs in the
 above-referred article. It includes ELISA results, protein phosphorylation
 assays, cell proliferation assays, flow cytometry analysis of T lymphocyte
 subpopulations, and anti-tumor  experiments in animal models. 
description: Graphpad
description: Molecular reshaping of phage-displayed Interleukin-2 at beta chain
 receptor interface to obtain potent super-agonists with improved
 developability profiles The dataset contains 19 original figures
 constructed with Graphpad (including the corresponding source data)
 presented in the above-referred manuscript. Authors: Gertrudis Rojas1\*,
 Ernesto Relova-Hernndez1, Annia Prez-Rivern1, Camila Castro-Martnez1,
 Osmany Diaz-Bravo1, Yanelys Cabrera Infante1, Tania Gmez1, Joaqun
 Solozbal1, Ana Beatriz DazBravo1, Maren Schubert2, Marlies Becker2,
 Beatriz Prez-Massn1, Dayana Prez-Martnez1, Rydell Alvarez-Arzola1, Osmany
 Guirola3, Glay Chinea3, Luis Graca4, Stefan Dbel2, Kalet Len1, Tania
 Carmenate1 Author affiliations: 1Center of Molecular Immunology, calle 216
 esq 15, apartado 16040, Atabey, Playa, CP 11300, La Habana, Cuba
 2Technische Universitt Braunschweig, Institute of Biochemistry,
 Biotechnology and Bioinformatics, Department of Biotechnology,
 Spielmannstrae 7, 38106 Braunschweig, Germany 3 Center of Genetic
 Engineering and Biotechnology, Ave 31 e/ 158 y 190, apartado 6162, Playa,
 CP 11300, La Habana, Cuba 4Instituto de Medicina Molecular Joo Lobo
 Antunes, Faculdade de Medicina da Universidade de Lisboa, Centro Acadmico
 de Medicina de Lisboa, Lisbon, Portugal \*Correspondence should be
 addressed to G.R. (grojas@cim.sld.cu) The contents of each file are
 detailed below: Figure 1a. Diversity of the soft-randomization human IL-2
 library. The library was constructed by Kunkel mutagenesis on a human IL-2
 gene-containing phagemid template, using two mutagenic spiked
 oligonucleotides which targeted beta interface segments 12-23 and 81-95.
 The composition of oligonucleotides was biased to the original wild-type
 sequence, but introduced limited variability at eight and seven codons of
 these segments, respectively. Library phages were panned on immobilized
 extracellular domain of human IL-2 receptor beta chain (three selection
 rounds). Deducing protein sequences of a sample of cytokine-displaying
 clones picked before and after selection revealed that the number of amino
 acid (aa) replacements per sequence increased upon selection. Figure 1b.
 Beta chain reactivity of phage pools selected from a soft-randomization
 human IL-2 library. The library was constructed by Kunkel mutagenesis on a
 phagemid template containing human IL-2 gene, using two mutagenic spiked
 oligonucleotides which targeted beta interface segments 12-23 and 81-95.
 The composition of oligonucleotides was biased to the original wild-type
 sequence, but introduced limited variability at eight and seven codons of
 these segments, respectively. Library phages were panned on immobilized
 extracellular domain (ECD) of human IL-2 receptor beta chain (three
 selection rounds). Beta chain reactivity of phage pools obtained before
 and after selection rounds was evaluated by ELISA on polyvinyl chloride
 microtitration plates coated with beta chain ECD. Bound phages were
 detected with an anti-M13 antibody conjugated to horseradish peroxidase.
 Phage-displayed versions of human IL-2 and H9 superkine were used as
 controls. Binding specificity was assessed with a non-related coating
 protein (bovine serum albumin). Each phage sample was evaluated twice on
 each coating protein. Bars indicate the mean absorbance values of both
 determinations, while dots represent individual data points. Figure 2e.
 Charge shift after phage selection from the secondary library S1. The
 library was constructed by Kunkel mutagenesis on a human IL-2
 gene-containing phagemid template, using a mutagenic oligonucleotide that
 introduced full randomization at positions 81, 83, 84 and 87, and limited
 variability (Pro/Ala) at position 82. Library phages were panned on
 immobilized extracellular domain of human IL-2 receptor beta chain (three
 rounds). Plasmid samples of cytokine-displaying clones from the unselected
 library and of those obtained after panning were sequenced, and protein
 sequences were deduced. Analysis of the net charge in the segment 81-87
 within both groups revealed a shift towards higher negative charges upon
 selection. Figure 2f. Beta chain reactivity of phage selected from the
 secondary library S1. The library was constructed by Kunkel mutagenesis on
 a human IL-2 gene-containing phagemid template, using a mutagenic
 oligonucleotide that introduced full randomization at positions 81, 83, 84
 and 87, and limited variability (Pro/Ala) at position 82. Library phages
 were panned on immobilized extracellular domain (ECD) of human IL-2
 receptor beta chain (three rounds). Reactivity of phage pools obtained
 before and after selection rounds was evaluated by ELISA on polyvinyl
 chloride microtitration plates coated with beta chain ECD. Bound phages
 were detected with an anti-M13 antibody conjugated to horseradish
 peroxidase Phage-displayed hIL-2 and H9 superkine were used as controls.
 Binding specificity was assessed with a non-related coating protein
 (bovine serum albumin). Each phage sample was evaluated twice on each
 coating protein. Bars indicate the mean absorbance values of both
 determinations, while dots represent individual data points. Figure 3g.
 Beta chain reactivity of phage clones selected from the secondary library
 S2. Positions 81, 83, 84 and 87 were totally randomized in S2 library and
 P82 was replaced by the mixture Pro/Ala. L80, L85, I86, I89, I92 and V93
 were substituted by a mixture of hydrophobic residues (Ile, Leu, Met, Phe
 and Val). Library was constructed by Kunkel mutagenesis on a human IL-2
 gene-containing phagemid template. Phages were panned on immobilized
 extracellular domain (ECD) of IL-2 receptor beta chain (three rounds).
 Thirteen different sequences were retrieved after selection. Reactivity of
 purified phage preparations from selected clones was evaluated by ELISA on
 polyvinyl chloride microtitration plates coated with either beta chain ECD
 or anti-c-myc tag 9E10 antibody. Bound phages were detected with an
 anti-M13 antibody conjugated to horseradish peroxidase. Relative
 reactivity was calculated as the ratio between signals obtained with beta
 chain and with 9E10. Phage-displayed hIL-2 and H9 superkine were used as
 controls. Bars represent relative reactivities. Figure 3h. Charge shift
 after phage selection from the secondary library S2. Positions 81, 83, 84
 and 87 were totally randomized in S2 library and P82 was replaced by the
 mixture Pro/Ala. L80, L85, I86, I89, I92 and V93 were substituted by a
 mixture of hydrophobic residues (Ile, Leu, Met, Phe and Val). Library was
 constructed by Kunkel mutagenesis on a human IL-2 gene-containing phagemid
 template. Phages were panned on immobilized extracellular domain of IL-2
 receptor beta chain (three rounds). Thirteen different sequences were
 retrieved after selection. Plasmid samples of cytokine-displaying clones
 from the unselected library and of those obtained after panning were
 sequenced, and protein sequences were deduced. Analysis of the net charge
 in the segment 81-87 within both groups revealed a shift towards higher
 negative charges upon selection. Figure 4a. Characterization of beta chain
 reactivity of recombinant proteins derived from S1 library screening.
 Recombinant proteins comprising the new human IL-2 variants designed upon
 S1 library screening, fused to a human IgG1 Fc region, were produced by
 transient transfection of HEK-293T cells adapted to grow in suspension,
 and purified by protein A affinity chromatography. Control fusion proteins
 derived from human IL-2 (K35E), from its single-mutated variant (R81D) and
 from the H9 superkine, were included. The reactivity of the purified
 proteins (31.25 ng/mL) was evaluated by ELISA on polyvinyl chloride
 microtitration plates coated with a recombinant fusion protein comprising
 human IL-2R beta subunit extracellular domain and mouse IgG2a Fc. Bound
 proteins were detected with an anti-human IgG antibody conjugated to
 horseradish peroxidase. Each protein sample was evaluated twice. Bars
 indicate the mean absorbance values of both determinations, while dots
 represent individual data points. Figure 5i. Characterization of thermal
 stability of recombinant proteins derived from S2 library screening.
 Recombinant proteins comprising the new human IL-2 variants designed upon
 S2 library screening, fused to a human IgG1 Fc region, were produced by
 transient transfection of HEK-293T cells adapted to grow in suspension,
 and purified by protein A affinity chromatography. Stability of the fusion
 proteins under thermal stress was assessed by ELISA after incubating them
 at 50C. Purified proteins (15.6 ng/mL) were incubated on polyvinyl
 chloride microtitration plates coated with the recombinant fusion protein
 comprising IL-2 receptor beta extracellular doman fused to a mouse IgG2a
 Fc domain. Bound proteins were detected with an anti-human IgG antibody
 conjugated to horseradish peroxidase. The fusion protein derived from from
 H9 superkine was included as control. Untreated proteins were also
 evaluated and used as references to calculate the relative reactivity (%)
 of each heated sample. Figure 6d. Beta chain reactivity of recombinant
 proteins derived from human IL-2 variants with mutations at position 92. A
 panel of recombinant proteins comprising human IL-2 (K35E) and H9
 superkine variants with or without additional replacements at position 92,
 fused to a human IgG1 Fc region, was produced by transient transfection of
 HEK-293T cells adapted to grow in suspension. The proteins were purified
 by protein A affinity chromatography. The reactivity of purified proteins
 was titrated by ELISA on polyvinyl chloride microplates coated with a
 recombinant fusion protein comprising IL-2R beta subunit extracellular
 domain and mouse IgG2a Fc. Bound proteins were detected with an anti-human
 IgG antibody conjugated to horseradish peroxidase. Each protein
 concentration was evaluated twice. Lines connect the mean absorbance
 values at different concentrations of the same protein. Symbols represent
 individual data points. Figure 8a. Capacity of IL-2-derived recombinant
 fusion proteins to induce CTLL-2 proliferation. Mouse CTLL-2 cells were
 grown during 48h in the presence of serial dilutions of recombinant
 Fc-fusion proteins comprising the new IL-2 beta super-binders derived from
 library screening. Reference proteins IL-2(K35E)/Fc and H9(K35E)/Fc were
 also included. Fc-fused IL-6 was used as negative control. Cells were
 stained with Alamar Blue dye and the proliferation curves were constructed
 by plotting the difference between absorbances at 540 and 620 nm as a
 function of fusion protein concentration. Two replicates of each protein
 concentration were analysed. Symbols represent the values of both
 determinations. Figure 8b. Capacity of IL-2-derived recombinant fusion
 proteins to induce CD25-KO CTLL-2 proliferation. CTLL-2 cells transduced
 with lentiviral particles encoding a sgRNA suitable to ablate expression
 of IL-2receptor alpha subunit gen (CD25-KO CTLL-2 model) were grown during
 48h in the presence of serial dilutions of recombinant Fc-fusion proteins
 comprising the new IL-2 beta super-binders derived from library screening.
 Reference proteins IL-2(K35E)/Fc and H9(K35E)/Fc were also included.
 Fc-fused IL-6 was used as negative control. Cells were stained with Alamar
 Blue dye and the proliferation curves were constructed by plotting the
 difference between absorbances at 540 and 620 nm as a function of fusion
 protein concentration. Two replicates of each protein concentration were
 analysed. Symbols represent the values of both determinations. Figure 8c.
 Capacity of IL-2-derived recombinant fusion proteins to induce STAT5
 phosphorylation in CTLL-2 cells. CTLL-2 cells were stimulated during 40
 min with the set of recombinant Fc-fusion proteins comprising the new IL-2
 beta super-binders derived from library screening, fixed, permeabilized
 and stained with an APC-labelled antibody against phosphorylated STAT5.
 The resulting fluorescence signals (determined by flow cytometry) measured
 signalling capacity of the proteins on the cells. Fc-fused IL-6 was used
 as negative control. Two replicates of each protein concentration were
 analysed. Symbols represent the values of both determinations. Figure 8d.
 Capacity of IL-2-derived recombinant fusion proteins to induce STAT5
 phosphorylation in CD25-KO CTLL-2 cells. CTLL-2 cells transduced with
 lentiviral particles encoding a sgRNA suitable to ablate expression of
 IL-2receptor alpha subunit gen (CD25-KO CTLL-2 model) were stimulated
 during 40 min with the set of recombinant Fc-fusion proteins comprising
 the new IL-2 beta super-binders derived from library screening, fixed,
 permeabilized and stained with an APC-labelled antibody against
 phosphorylated STAT5. Fc-fused IL-6 was used as negative control. The
 resulting fluorescence signals (determined by flow cytometry) measured
 signalling capacity of the proteins on the cells. Two replicates of each
 protein concentration were analysed. Symbols represent the values of both
 determinations. Figure 9b. Increase in the spleen cell content induced by
 IL-2-derived fusion proteins. Four groups of five mice each received daily
 injections (during four days) of recombinant proteins comprising
 IL-2-derived beta super-binders fused to human Fc(LALA). Two additional
 groups were treated with similarly formatted proteins containing either
 wild-type human IL-2(K35E) or H9 superkine. Mice from a control group
 received PBS injections. All the animals were sacrificed at the fifth day,
 and their spleens were collected and macerated for cell count. Dots
 indicate spleen cell content of individual animals. Lines represent mean
 values and error bars indicate SD within each group of five animals
 receiving the same treatment. Figure 9d. In vivo expansion of effector T
 cell population CD8+CD44hiCD122hi induced by IL-2-derived fusion proteins.
 Four groups of five mice each received daily injections (during four days)
 of recombinant proteins comprising IL-2-derived beta super-binders fused
 to human Fc(LALA). Two additional groups were treated with similarly
 formatted proteins containing either wild-type human IL-2(K35E) or H9
 superkine. Mice from a control group received PBS injections. All the
 animals were sacrificed at the fifth day, and their spleens were collected
 and macerated for characterization by flow cytometry. Dots indicate total
 numbers of CD8+CD44hiCD122hi T cells in individual animals Lines represent
 mean values and error bars indicate SD within each group of five animals
 receiving the same treatment. Figure 9f. In vivo induction of effector
 cell proliferation induced by IL-2-derived fusion proteins. Four groups of
 five mice each received daily injections (during four days) of recombinant
 proteins comprising IL-2-derived beta super-binders fused to human
 Fc(LALA). Two additional groups were treated with similarly formatted
 proteins containing either human IL-2 (K35E) or H9 superkine. Mice from a
 control group received PBS injections. All the animals were sacrificed at
 the fifth day, and their spleens were collected and macerated for cell
 characterization by flow cytometry. Dots indicate the number of
 proliferating effector cells (CD8+Ki67+) of individual animals. Lines
 represent mean values and error bars indicate SD within each group of five
 animals receiving the same treatment. Figure 9h. In vivo expansion of T
 regulatory cells induced by IL-2-derived fusion proteins. Four groups of
 five mice each received daily injections (during four days) of recombinant
 proteins comprising IL-2-derived beta super-binders fused to human
 Fc(LALA). Two additional groups were treated with similarly formatted
 proteins containing either human IL-2 (K35E) or H9 superkine. Mice from a
 control group received PBS injections. All the animals were sacrificed at
 the fifth day, and their spleens were collected and macerated for
 characterization by flow cytometry. Dots indicate the total numbers of
 Treg cells (CD4+CD25+Foxp3+) of each individual animal. Lines represent
 mean values and error bars indicate SD within each group of five animals
 receiving the same treatment. Figure 10b. Figure 10. Anti-metastatic
 effects of IL-2-derived fusion proteins. Four groups of seven C57BL/6 mice
 each were inoculated intravenously with 1 x 105 MB16F0 melanoma cells, and
 subsequently treated with five daily injections of the recombinant
 proteins comprising human IL-2-derived beta super-binders fused to human
 Fc(LALA). Two additional groups were treated with similarly formatted
 proteins containing either human IL-2(K35E) or H9 superkine. Mice from a
 control group received PBS injections. All the animals were sacrificed at
 day 21, and their lungs were extracted for counting the number of
 metastatic nodules. Two animals, from the groups treated with PBS and
 IL-2(K35E)Fc(LALA) respectively, died before completion of the experiment
 and were excluded. Lines represent mean values and SD within each group.
 Figure 10c. Figure 10. Anti-tumour effects of IL=2=derived fusion
 proteins. Seven groups of five BALB/c mice each were inoculated
 subcutaneously with 1 x 105 CT26 cells in the left flank, and subsequently
 treated with five daily injections of the set of recombinant proteins
 comprising human IL-2-derived beta super-binders, wild-type IL-2 (K35E)
 and H9 superkine fused to human Fc(LALA), and with PBS in the case of the
 control group. After tumours became palpable, their length and width were
 measured every other day in order to calculate tumor volume until one
 tumor side reached 18 mm. Tumor volumes are represented in c. Lines
 represent mean values and SD within each group