Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells

Abstract

Background and purpose: In 2006, a life-threatening 'cytokine storm', not predicted by pre-clinical safety testing, rapidly occurred in all six healthy volunteers during the phase I clinical trial of the CD28 superagonist monoclonal antibody (mAb) TGN1412. To date, no unequivocal explanation for the failure of TGN1412 to stimulate profound cytokine release in vitro or in vivo in species used for pre-clinical safety testing has been established. Here, we have identified a species difference almost certainly responsible for this disparate immunopharmacology.

Experimental approach: Polychromatic flow cytometry and intracellular cytokine staining were employed to dissect the in vitro immunopharmacology of TGN1412 and other therapeutic mAbs at the cellular level to identify differences between humans and species used for pre-clinical safety testing.

Key results: In vitro IL-2 and IFN-γ release from CD4+ effector memory T-cells were key indicators of a TGN1412-type response. This mechanism of cytokine release differed from that of other therapeutic mAbs, which can cause adverse reactions, because these other mAbs stimulate cytokine release primarily from natural killer cells. In contrast to humans, CD28 is not expressed on the CD4+ effector memory T-cells of all species used for pre-clinical safety testing, so cannot be stimulated by TGN1412.

Conclusions and implications: It is likely that activation of CD4+ effector memory T-cells by TGN1412 was responsible for the cytokine storm. Lack of CD28 expression on the CD4+ effector memory T-cells of species used for pre-clinical safety testing of TGN1412 offers an explanation for the failure to predict a 'cytokine storm' in humans.

Figure 1

Kinetic analysis of cytokine-producing human PBMCs stimulated in vitro with 1 µg per well Fc-immobilized TGN1412 (IgG4κ), immobilized TGN1412 (IgG4κ), immobilized CD28 superagonist ANC28.1 (murine IgG1), immobilized CD28 agonist CD28.2 (murine IgG1) or immobilized isotype-matched control (IgG4κ). The frequency of cytokine positive cells is expressed as a percentage of total lymphocytes for (A) TNF-α, (B) IFN-γ and (C) IL-2, against time in minutes. Data shown are the mean responses of eight donors obtained from two independent experiments.

Figure 2

Cytokine release from human PBMCs stimulated in vitro for 24, 48 and 72 h with 1 µg per well immobilized isotype-matched control (IgG4κ), Mabthera (IgG1), Campath-1H (IgG1) and TGN1412 (IgG4κ). The lectin PHA at 10 µg·mL^–1 was used as positive control. Cytokine release was measured by elisa of culture supernatants. (A) TNF-α release, (B) IFN-γ release, (C) IL-2 release and (D) IL-8 release are expressed in pg·mL^–1. Data shown are mean responses of nine donors obtained from two independent experiments.

Figure 3

Intracellular cytokine staining of human PBMCs for (A) IL-2 and (B) TNF-α. Prior to staining, PBMCs were stimulated with 1 µg per well immobilized isotype-matched control (IgG4κ), Mabthera (IgG1), Campath-1H (IgG1) or TGN1412 (IgG4κ) for 360 min. PBMCs were counter-stained for surface CD4 expression. The frequency of cytokine positive cells is expressed as a percentage of total lymphocytes. Cells in the right-hand quadrants are positive for cytokine expression. Cells in the upper quadrants are positive for CD4 expression. The mitogens PMA + ionomycin were used as positive control. Data from two representative donors are shown (similar data were obtained with 14 additional donors from three independent experiments).

Figure 5

Intracellular cytokine staining of human CD4⁺ effector memory T-cell (Tem)-depleted (A) and -enriched (B) PBMCs for TNF-α, IL-2 and IFN-γ, following stimulation with 1 µg per well immobilized TGN1412. Tem-depleted (left panels) and Tem-enriched (right panels) PBMCs were surface stained for T-cell markers and intracellular cytokines; analysis shown is gated on CD4⁺ T-cells. Cells in the right-hand quadrants are positive for TNF-α, cells in the upper quadrants are positive for IL-2, cells shown in red are positive for IFN-γ and cells shown in green are negative for IFN-γ. Cells in the upper right corner in red are positive for TNF-α, IL-2 and IFN-γ. The frequency of cytokine positive cells is expressed as a percentage of CD4⁺ T-cells. Data from two representative donors are shown (similar data were obtained with four additional donors from two independent experiments).

Figure 4

Intracellular cytokine staining of human CD4⁺ memory T-cells for (A) TNF-α, (B) IFN-γ and (C) IL-2, following stimulation with 1 µg per well immobilized TGN1412. Analysis shown is gated on CD4⁺ T-cells. Cells in the upper quadrants are CD45RO⁺ memory T-cells, and cells in the lower quadrants are CD45RO^- naive T-cells. Cells in the right-hand quadrants are positive for the indicated cytokines. The frequency of cytokine positive cells is expressed as a percentage of CD4⁺ T-cells. Data from two representative donors are shown (similar data were obtained with six additional donors from two independent experiments).

Figure 6

Comparative immunophenotyping of human, cynomolgus and rhesus macaque CD4⁺ T-cells. All analysis shown is gated on CD4⁺ T-cells. (A) Analysis of memory markers using CD28 and CCR7 staining. Human, cynomolgus and rhesus macaque naive CD4⁺ T-cells (CCR7⁺CD28⁺) and central memory T-cells (CCR7⁺CD28⁺), macaque transitional memory T-cells (CCR7^-CD28⁺) and human effector memory CD4⁺ T-cells (CCR7^-CD28⁺) all express CD28. In contrast, both cynomolgus and rhesus effector memory T-cells (CCR7^-CD28^-) do not express CD28. Data from two representative donors are shown (similar data were obtained with two additional rhesus, two cynomolgus and six human donors from two independent experiments). (B) Intracellular IFN-γ staining versus CD28 expression on CD4⁺ T-cells following PMA and ionomycin stimulation for 4 h. Human CD4⁺ T-cells constitutively express CD28, so IFN-γ staining stems only from the CD28⁺ population. Macaque CD4⁺ T-cells can be subdivided into CD28⁺ (naive, central and transitional memory T-cell) and CD28^- (effector memory T-cell) populations, both of which produce IFN-γ following PMA and ionomycin stimulation for 4 h. A greater fraction of macaque CD4⁺CD28^- T-cells produce IFN-γ compared to CD4⁺CD28⁺ T-cells following mitogen stimulation (similar data were obtained with two additional rhesus, two cynomolgus and two human donors).