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    Immunoengineering

    (J-380) Immunomodulation for tissue repair through affinity-based binding partners

    Friday, October 13, 2023
    3:30 PM – 4:30 PM PDT
    Location: Exhibit Hall - Row J - Poster # 380
    Introduction:: Traumatic injuries can result in the down-regulation of proteins involved in the wound healing cascade, leading to diminished tissue regeneration[1]. Previous studies have demonstrated that supplementing immunomodulatory proteins can help to reestablish functional healing[2]. However, these proteins must be delivered to the injury site with sufficient spatiotemporal control to maximize therapeutic impact while minimizing off-target effects. To address this challenge, our lab uses affinity interactions in order to control protein delivery with bioengineered binding partners called affibodies[3]. Affibodies are small alpha-helical proteins with variable residues that can be mutated to confer the binding affinity and specificity for various other proteins. The goal of this study was to use affibodies, coupled with hydrogel delivery vehicles, to control the delivery of interleukin-4 (IL-4), a cytokine associated with the transition from the pro-inflammatory to the anti-inflammatory stage of wound healing[4].

    Materials and Methods:: Prospective IL-4-binding affibody sequences were isolated from a yeast surface display library of 108 affibody variants using fluorescence- and magnetic-activated cell sorting. For recombinant expression, affibody sequences, modified with a C-terminal hexahistidine tag and a cysteine residue for future conjugation, were ligated into pET28b+ vectors and transformed into BL21 E. coli cells for protein expression. The expressed affibodies were purified using immobilized metal affinity chromatography. Soluble purified affibody was characterized through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD) to confirm appropriate size and protein folding. Biolayer interferometry (BLI) was used to identify the strength of binding of each affibody to IL-4, denoted as a dissociation constant, representing the rate of dissociation over the rate of association. Streptavidin-functionalized probes were loaded with 100nM of biotinylated IL-4 and introduced to a range of affibody concentrations (25nM-1nM). Curve-fitting of the association and dissociation wavelength shifts was used to obtain the dissociation constant for each affibody. BLI on IL-4 affibodies was also performed using bone morphogenetic protein-2 (BMP-2) and granulocyte macrophage colony stimulating factor (GM-CSF) to confirm specificity of each affibody to IL-4. IL-4-specific affibodies were conjugated to 20kDa, 4-arm polyethylene glycol-maleimide (PEG-Mal) via a thiol-maleimide reaction[3]. The solution was crosslinked with dithiothreitol (DTT) to form IL-4-affibody conjugated hydrogels[5]. The hydrogels were loaded with 100ng IL-4, then were allowed to release the IL-4 over 28 days. IL-4 encapsulation and release timepoints were collected from the supernatant of the hydrogels and quantified using an IL-4 enzyme-linked immunosorbent assay (ELISA).

    Results, Conclusions, and Discussions:: Three prospective affibody sequences were identified from yeast surface display, and subsequently collected in soluble form following E. coli transformation. SDS-PAGE indicated that each affibody had the expected mass of ~7 kDa. CD of each affibody received two troughs at 222 and 208nm and a peak at 193nm, which is characteristic of an alpha-helical structure, indicating appropriate affibody folding (Fig. 1A). BLI measurements demonstrated that the first affibody has a dissociation constant of 92,000nM, which is characteristic of a low affinity for IL-4. The second affibody was found to have a dissociation constant of 2.72nM, which is characteristic of a high affinity for IL-4 (Fig. 1B). The high affinity IL-4 affibody did not demonstrate binding to BMP-2 and GM-CSF, suggesting high specificity to IL-4. The low affinity affibody did not bind to BMP-2, but demonstrated a slight affinity for GM-CSF, with a dissociation constant of 12,500nM, suggesting only moderate specificity to IL-4. Affibody-conjugated hydrogels for each affibody demonstrated comparable encapsulation of IL-4, compared to hydrogels without affibodies (Fig. 1C). Affibody conjugated hydrogels also released IL-4 significantly slower compared to the hydrogels with no affibodies (Fig. 1D).

    We have demonstrated that the delivery of IL-4 can be controlled through the use of unique affibody binding partners in PEG-Mal hydrogels. The high and low affinity affibodies indicate that protein release is tunable through the affinity-interactions between the affibody and IL-4. In the future, we will assess the effects of affibody-mediated IL-4 release on its immunomodulatory properties by quantifying the expression of anti-inflammatory genes in macrophages differentiated from THP-1 monocytes[6]. Ultimately, IL-4 delivery to severe tissue injuries may be controlled by IL-4-specific affibodies as a mechanism for reestablishing functional anti-inflammatory immune responses. 


    Acknowledgements (Optional): :

    References (Optional): :

    [1] Dorogin, et al. Biomater. Sci. 9: 2339-2361 (2021). 


    [2] Sadtler, et al. Science. 352: 66-370 (2016).


    [3] Dorogin, et al. Adv Healthc Mater. (2023). 


    [4] Luzina, et al. J Leukoc Biol. 92: 753-764(2012).


    [5] García. Ann Biomed Eng. 42: 312-322(2013).


    [6] Genin, et al. BMC Cancer. 15:577 (2015).