Insufficient (Sub-native) Helix Content in Soluble/Solid Aggregates of Recombinant and Engineered Forms of IL-2 Throws Light on How Aggregated IL-2 is Biologically Active

Abstract

Interleukin 2 (IL-2) is an extremely aggregation-prone, all-alpha helical cytokine. In its receptor-bound state, ~72 % of the polypeptide chain adopts helical structure and there is no beta sheet content whatsoever. In the past, recombinant IL-2 has been formulated and used therapeutically in humans, following production in E. coli. Therapeutic IL-2 consists entirely of functionally-active soluble aggregates with ~30 subunits per aggregate particle. Side-effects attributed to aggregation resulted in discontinuation of usage over a decade ago. Structurally, and biochemically, activity in IL-2 aggregates can potentially be explained in one of two ways : (a) individual IL-2 chains exist in sterically-accessible, receptor binding-competent (native) structures, allowing aggregates to bind directly to IL-2 receptors (IL-2R); alternatively, (b) IL-2 chains dissociate from aggregates, become free to adopt native structure, and then bind to IL-2R. We produced native IL-2 and numerous engineered forms in E. coli with the objective of obtaining insights into these possibilities. Each IL-2 variant was subjected to size exclusion chromatography, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). All forms produced and studied (including those with native IL-2 sequences) turned out to aggregate and also display less than ~50 % helix content as well as significant beta sheet content. No conditions were found that obviate aggregation. Aggregated IL-2 is thus insufficiently native-like to bind to IL-2R. Activity in aggregates thus probably owes to adoption of receptor binding-competent structures by chains that have already dissociated from aggregates.

Directed evolution of super-secreted variants from phage-displayed human Interleukin-2

“…The discovery of a simple structural solution (single mutation-based) reducing the remarkable aggregation propensity of IL-2 and improving its secretion contrasts with the failure of previous attempts to do so. Neither the introduction of N - and C -terminal fusions and artificial disulphide bonds nor the mutational disruption of the negatively charged cavity at tail produced the expected results, leading to the view that IL-2 aggregation propensity is too high to be conquered through engineering 23 . Changing the charge near the cleavage site between the signal peptide and the N -terminus of IL-2 (replacing K8/K9 by acidic residues) did not result in periplasmic secretion in E. coli host 42 .…”

“…The effects of K35E can be linked to the change in the local net charge. Ionic interactions between positively charged protrusions at the nose side of the IL-2 cylinder and negatively charged cavities at the tail have been postulated to mediate nose-tail docking of IL-2 molecules leading to the formation of polymeric aggregates 23 . K35E disrupts the charge distribution at the positive protruding end (K32, K35, R38, K76) and could thus affect aggregation (Fig.…”

“…Alternative expression systems, based on yeast, insect and mammalian cells have also been explored 22 . Regardless of the expression system, IL-2 and other structurally related alpha-helical cytokines have a very strong aggregation propensity, resulting in the formation of aggregates of up to 30 molecules even in optimized formulations 23,24 . A link between aggregation, toxicity and undesired immunogenicity of IL-2 has been postulated to compromise its therapeutic usefulness 25,26 .…”

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography

“…In bacteria, in which the recombinant proteins are in general not secreted to the media, aggregation of the recombinant protein results in large cytoplasmic structures called inclusion bodies (IBs) [10]. Soluble aggregates, probably intermediates in IB formation, also abound [11][12][13], since aggregation of recombinant proteins is a complex event that involves a wide spectrum of conformational conformers [14][15][16][17], ranging from properly folded versions to misfolded, amyloidal forms [16]. Globally, the initial step in recombinant protein purification is the separation, after cell lysis, of the soluble cell fraction from the insoluble cell fraction.…”

Generation of IL‐2‐Fc‐antibody conjugates by click chemistry

“…[8] On the other hand, IL-2-Fc fusion proteins have a high degree of protein aggregation. [9,10] IL-2-based aggregation can cause significant toxicity and immunogenicity. [11,12] Recently the IL-2-Fc-based aggregation problem was addressed with the charge reversal point mutation K35E in the human IL-2 portion of the fusion protein.…”