Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses

Abstract

Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133), can enhance the in vitro innate immune response of a population of naive human peripheral blood mononuclear cells. This response depended on the aggregate type, inherent immunogenicity of the monomer, and donor responsiveness, and required a high number of particles, well above that detected in marketed drug products, at least in this in vitro system. We propose a cytokine signature as a potential biomarker of the in vitro peripheral blood mononuclear cell response to aggregates. The cytokines include IL-1β, IL-6, IL-10, MCP-1, MIP-1α, MIP-1β, MMP-2, and TNF-α. IL-6 and IL-10 might have an immunosuppressive effect on the long term immune response. Aggregates made by stirring induced the highest response compared with aggregates made by other methods. Particle size in the 2-10 μm range and the retention of some folded structure were associated with an increased response. The mechanism of aggregate activation at the innate phase was found to occur through specific cell surface receptors (the toll-like receptors TLR-2 and TLR-4, FcγRs, and the complement system). The innate signal was shown to progress to an adaptive T-cell response characterized by T-cell proliferation and secretion of T-cell cytokines. Investigating the ability of aggregates to induce cytokine signatures as biomarkers of immune responses is essential for determining their risk of immunogenicity.

FIGURE 1.

Representative cytokine profiles of a population of PBMC in response to monomeric or aggregated antibodies. A, cytokine expression profile of PBMC from one representative responding donor treated with either monomeric or aggregated mAb1. Multiplex cytokine analysis was used to detect the amount (pg/ml) of 47 cytokines released as an innate response. Vertical black lines represent cytokines that were found to increase in the presence of the aggregated mAbs above the monomeric mAb or background (SI ≥2.0) for this donor. B, heat map illustration of statistically significant increases in cytokine release from PBMC (15–22 donors) at the innate phase (20 h) treated with monomeric mAb1, mAb2, or mAb3. A subset of the 47 cytokines tested is shown, where the remaining cytokines were not impacted. p values generated by a mixed effect statistical model were used to indicate statistically significant differences between donors treated with each monomeric mAb and the background response. Cytokine abbreviations are as follows: A1AT, α₁-antitrypsin; A2M, α₂-macroglobulin; B2M, β₂-microglobulin; BDNF, brain-derived neurotrophic factor; C3, complement 3; CRP, C reactive protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; Hp, haptoglobin; ICAM-1, intercellular adhesion molecule-1; IFN-γ, interferon-γ; IL-1α, -1β, -1ra, -2, -3, -4, -5, -6, -7, -8, -10, -12p40, -12p70, -15, -17, -18, -23, interleukin-1α, -1β, -1 receptor antagonist, -2, -3, -4, -5, -6, -7, -8, -10, -12p40, -12p70, -15, -17, -18, and -23; MCP-1, monocyte chemotactic protein-1; MIP-1α, -1β, macrophage inflammatory protein-1α, -1β; MMP-2, -3, -9, matrix metalloproteinase-2, -3, -9; SCF, stem cell factor; TIMP-1, tissue inhibitor of metalloproteinase-1; TNF-α, -β, -RII, tumor necrosis factor-α, -β, -receptor II; VCAM-1, vascular cell adhesion molecule-1; VDBP, vitamin D-binding protein; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.

FIGURE 2.

Aggregation of biotherapeutics enhances the innate response of PBMC. PBMC from 22 donors were challenged with either monomeric or aggregated mAbs and tested for the release of signature cytokines by multiplex cytokine analysis at the innate phase (20 h). A, average SI of positive donors (SI ≥2.0, colored bars) and percentage of donors that responded (% donors, gray bars) to the aggregated mAb above the monomeric mAb is shown for mAb1, mAb2, and mAb3. Black dots depict responding individuals and highlight the distribution of responses across the population tested. These population bar graphs highlight the response of individuals to capture all events. Different aggregate types are depicted horizontally as follows: stir-20h (red), stir-3d (orange), 65C/pH 8.5 (green), and syringe-so⁺ (blue). B, heat maps of statistically significant increases in the cytokine signature above the monomeric mAb (top panel) or background (bottom panel) are shown for mAb1, mAb2, and mAb3. These panels reflect the average response of the population. p values generated by a mixed effect statistical model were used to indicate statistically significant differences between donors treated with the aggregated mAbs and the same donors treated with either the monomeric mAb or the background response.

FIGURE 3.

High particle numbers correlate with and are required for aggregate activation. PBMC from eight donors were challenged with either mAb1-microspheres or mAb1 aggregates and assessed for cytokine secretion by multiplex cytokine analysis at the innate phase (20 h). A, number of particles in the mAb1-microspheres and aggregated samples were determined by light obscuration. Responses were compared between samples that had either an equal concentration of the mAb1 protein or equal particle numbers. B, average dose response of two donors to mAb1-microspheres. The average SI above background and standard deviation representing the variability of the population tested are shown. The number of particles at each concentration of mAb1-microspheres tested is shown, where the black arrow indicates the approximate threshold of particles for activation. C and D, average SI of positive donors (SI ≥2.0, colored bars) and percentage of donors that responded (% donors, gray bars) to mAb1-microspheres and mAb1 aggregates above monomeric mAb1. Black dots depict responding individuals.

FIGURE 4.

mAb1-microspheres retain a high percentage of their mAb1 folded structure. A, overlaid second derivative FTIR spectra of the monomeric mAb1 (black line), aggregated mAb1 pellet fractions (red, orange, and green lines), and mAb1-microspheres (purple line) show varying degrees of structural changes. B, % correlation coefficient (CC) was calculated between the spectra of the monomeric protein and the aggregated samples or mAb1-microspheres.

FIGURE 5.

Mechanism of antibody aggregate activation is mediated by cell surface receptors. A, PBMC were challenged with stir-20h aggregates in the absence and presence of inhibitory antibodies of cell surface receptors (FcγRI, FcγRII, FcγRIII, TLR-2, and TLR-4) or stir-20h aggregates that had been opsonized with catalytically active C3b or heat-inactivated C3b (56 °C). Supernatants were assessed by multiplex cytokine analysis at the innate phase (20 h). Cytokine release was calculated as a percentage of the total response to stir-20h aggregates in the absence of inhibitory antibodies or heat inactivation. Values shown represent the average of duplicate tests from one representative donor. B, percentage of donors (four donors tested) that were inhibited from responding to stir-20h aggregates by blocking antibodies or heat inactivation.

FIGURE 6.

Average CD4⁺ T-cell response from 50 individuals was enhanced when challenged with aggregated biotherapeutic. The CD4⁺ T-cell response from 50 healthy donors to mAb2 monomeric and stir-20h samples was assessed at the adaptive phase (up to 8 days) using T-cell proliferation assays ([³H]thymidine uptake) (A) and IL-2 cytokine secretion (ELISPOT) (B). C, percentage of donors that responded positively in both the T-cell proliferation and IL-2 ELISPOT assays. The average SI of positive donors (SI ≥1.9) above the background response and the percentage of donors that responded are shown. D, supernatants were assessed for T-cell effector cytokines by multiplex cytokine analysis. The average SI of positive donors (SI ≥1.9) above the response to the monomeric mAb is shown. Black dots depict responding individuals.