Activation of human T cells by FcR nonbinding anti-CD3 mAb, hOKT3gamma1(Ala-Ala)

Abstract

Dimeric Fc receptor (FcR) nonbinding anti-CD3 antibodies have been developed to minimize toxicities associated with classical anti-CD3 monoclonal antibodies (e.g., OKT3). Studies with murine analogs of non-FcR-binding antibodies have shown reduced mitogenicity compared to OKT3. In a trial of an FcR nonbinding humanized anti-CD3 mAb hOKT3gamma1(Ala-Ala) for treatment of patients with type 1 diabetes, we found significant increases in IL-10 and IL-5 in the serum of 63% and 72% of patients, respectively, and TNF-alpha and IL-6 levels that were lower than those previously reported following OKT3 therapy. The activation signal delivered by hOKT3gamma1(Ala-Ala) was associated with calcium signaling and cytokine production by previously activated human cells in vitro. However, the production of IL-10, compared to IFN-gamma on a molar basis, was greater after culture with hOKT3gamma1(Ala-Ala) than with OKT3. Flow cytometric studies confirmed that OKT3 induced IFN-gamma and IL-10 production, but hOKT3gamma1(Ala-Ala) induced only detectable IL-10 production in CD45RO(+) cells. Moreover, in vivo, we found IL-10(+)CD4(+) T cells after drug treatment. These cells were heterogeneous but generally CD45RO(+), CTLA-4(-), and expressed CCR4. A subgroup of these cells expressed TGF-beta. Thus, the non-FcR binding anti-CD3 mAb, hOKT3gamma1(Ala-Ala) delivers an activation signal to T cells that is quantitatively and qualitatively different from OKT3. It leads to the generation of T cells that might inhibit the autoimmune response and may be involved in the beneficial effect on beta cell destruction in Type 1 diabetes.

Figure 1

Cytokine levels following administration of hOKT3γ1(Ala-Ala). The levels of cytokines in serum were measured as described in Methods. The average (± SEM) of the highest level of cytokines following drug administration on days 1, 2, 5, and 6 (first 12 patients) or days 1, 2, 3, and 4 (patients 9–20) for each patient in the two dosing protocols (see Methods) are shown. ptl, protocol.

Figure 2

Expression of CD25 and CD69 on peripheral T cells in patients receiving hOKT3γ1(Ala-Ala). The percentage of CD4⁺ and CD8⁺ cells expressing CD25 and CD69 are shown before, during, and after treatment with hOKT3γ1(Ala-Ala). There was a significant increase in the percentage of CD4⁺CD25⁺ (P = 0.0015), CD4⁺CD69⁺ (P < 0.0001), CD8⁺CD25⁺ (P = 0.0001), and CD8⁺CD69⁺ (P < 0.0005) T cells at day 7 (for the first 12 patients) or day 8 (for patients 9–20).

Figure 3

Downmodulation of CD62L on CD4^+/–CD25^+/– T cells during treatment with hOKT3γ1(Ala-Ala). PBMCs were isolated from patients before and after treatment with hOKT3γ1(Ala-Ala). The cells were stained with mAb’s to CD4, CD25, and CD62L, or isotype controls. Electronic gates were placed around CD4⁺ lymphocytes, and the percentage of the CD4⁺ cells staining with CD25 (left panels) or CD62L (right panels) are shown. These data are from a single patient, representative of four of five patients in whom this analysis was performed. The expression of CD62L was reduced on the CD4⁺CD25⁺ population of cells.

Figure 4

Density plot of calcium flux in recently activated T cells cultured with OKT3 or hOKT3γ1(Ala-Ala). An aliquot of cells that had been cultured with PHA followed by IL-2 were loaded with indo-1 and placed in culture (at 37°C) in HBSS alone, followed by OKT3 (top) or hOKT3γ1(Ala-Ala) (bottom) (1 μg/ml) with the addition of RAM at the indicated times (in seconds). The ratio of FL5/FL6, indicates intracellular calcium levels in individual cells, where an increased ratio reflects increased intracellular calcium. The addition of either hOKT3γ1(Ala-Ala) or OKT3 followed by cross-linking Ab led to increase in intracellular calcium.

Figure 5

Induction of cytokines by anti-CD3 mAb’s. CD45RO⁺- and CD45RO^–-enriched T cells were separated with paramagnetic beads and cultured with either OKT3 (10 μg/ml) or hOKT3γ1(Ala-Ala) (10 μg/ml) with anti-CD28 (1 μg/ml) in wells coated with RAM antibody (10 μg/ml) for 6 hours in the presence of monensin. The cells were then stained with mAb’s to CD45RO and CD3, fixed and permeabilized, and stained for intracellular cytokines as described in Methods, then analyzed by flow cytometry. For analysis, electronic gates were placed around the CD3⁺CD45RO⁺ or CD3^–CD45RO^– subsets.The percentage of gated cells staining for each cytokine are shown in the upper-right corner of each dot plot.

Figure 6

Expression of IL-10 in CD3^+/– T cells after treatment with hOKT3γ1(Ala-Ala). (a) PBMCs were isolated from a patient before and 1 week after the last dose of drug. The cells were cultured for 6 hours in the presence of monensin and CD28 and then stained for CD3 on the cell surfaces and intracellular IFN-γ (x axis) and IL-10 (y axis) after permeabilization. Further studies identified the IL-10⁺ cells as being CD4⁺ (see text). (b) The presence of IL-10⁺CD4⁺ T cells was studied before, during, and after treatment with the anti-CD3 mAb. The box indicates the time that the anti-CD3 mAb was given. The data represent the percentage of CD4⁺ T cells that are IL-10⁺ over time in the five of six patients in whom these cells were identified. (c) The IL-10⁺CD4⁺ T cells express CCR4 on their surfaces. The histogram shows surface staining with CCR4 on IL-10⁺CD4⁺ (solid line), IL-10^–CD4⁺ (dashed line), or background staining on CD4⁺ cells with an isotype control Ab (dotted line).