Oral Administration of OKT3 Monoclonal Antibody to Human Subjects Induces a Dose-Dependent Immunologic Effect in T Cells and Dendritic Cells

Patient population

Healthy males (≥18 years) not on therapy for medical or other illnesses were enrolled in accordance with the guidelines of the Hebrew University-Hadassah Institutional Committee for Human Clinical Trials, and the approval of the Israel Ministry of Health Committee for Human Trials. Of 35 potential study subjects screened, 18 met inclusion and exclusion criteria and were randomized to one of the six treatment groups.

OKT3 and β-Glucosylceramide

OKT3 (Orthoclone OKT-3) was purchased from Ortho Biotech Inc. (New Jersey, USA) and GC from Avanti Polar Lipids, Inc., Alabaster, Alabama, USA.

Drug administration

Nine subjects (three per group) received 0.2 mg, 1.0 mg or 5.0 mg of oral OKT3 daily for 5 days. Six subjects (three per group) received 7.5 mg of beta-glucosylceramide in combination with 0.2 mg or 1.0 mg of OKT3 and three subjects received GC alone. Immune parameters were measured on days 5, 10, and 30 (Fig. 1). All subjects were treated with 20 mg of omeprazole (a proton pump inhibitor) during the 5 days of dosing. Dosing occurred in the morning before breakfast at the study site following an 8-h fast. We administered a proton pump inhibitor to protect the antibody from the acidic environment of the gut.

Clinical and laboratory follow-up

All patients underwent a full medical history and physical examination, including review of adverse effects on days 1, 5, 10, and 30, along with complete blood counts, differential, electrolytes, liver and kidney function tests, lipid profile, C-reactive protein, and sedimentation rates. Antibodies to OKT3 were evaluated on day 30 using the human anti-mouse antibodies (HAMA)-ELISA kit (MEDAC, Hamburg, Germany)

1. Standardization of assays in untreated subjects

for the purpose of standardization, and in order to establish that immune changes we observed were related to treatment with oral anti-CD3 and not related to fluctuations in the assays, we performed the same assays that were used to measure immune function in treated subjects in untreated healthy controls longitudinally on days 0, 5, and 10. We observed no changes in any of the immune assays over the three time points measured, thus allowing us to ascribe immune changes observed in subjects given oral anti-CD3 as being related to treatment. An example for proliferation, 17 production and cell surface staining is shown in Fig. 2.

2. Antibodies and reagents

CD16/CD32-specific (FcBlock); FITC, PE, or APC-conjugated CD4-specific (L3T4); and, PE-conjugated CD25-specific (PC61) were purchased from BD PharMingen (San Jose, CA, USA). Affinity-purified biotinylated goat anti-LAP specific polyclonal antibody was purchased from R&D Systems (Minneapolis, MN, USA), and strepavidin-APC was used as secondary reagent for detecting the biotinylated primary antibody (R&D). 7-AAD for staining dead cells was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor-647 FOXP3 kit was purchased from BioLegend (San Diego, CA, USA). FITC-conjugated CD4 or CD8, PE-conjugated CD25 and their compensation controls, were from eBioscience (San Diego, CA, USA). Other markers as APC-conjugated CD11c and PE-conjugated CD40, CD80, CD83, CD86, and HLA-DR were also from eBioscience. FITC-conjugated Lin1 and APC-conjugated CTLA4 was purchased from BD PharMingen (San Jose, CA, USA).

3. Proliferation and cytokine assays

PBMCs were isolated from blood samples using Ficoll Hypaque solution and 2×10⁵ cells per well were cultured in triplicate in RPMI 1640 medium with 5% FBS (Biological Industries, Israel), 100 units/ml penicillin, 100 μg/ml streptomycin, 1% glutamine, 1% non-essential fatty acids, 1% sodium pyruvate and β-mercapto ethanol (Biological Industries, Israel). Cells were stimulated with 5 μg/ml soluble anti-CD3 mAb (eBioscience, CA, USA). For TGF-β, 2×10⁵ cells per well were cultured in serum free media (Biotarget, Biological Industries, Israel). Supernatants were collected at 48 h. Enzyme-linked immunosorbent assays for IL-13, IL-17, IFN-γ, and TGF-β were performed according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN). After collecting the supernatants, 1 μCi [³H]thymidine (Amersham Biosciences, UK) was added to each well, and cells were harvested 18 h later. Proliferation was measured by scintillation counting.

4. FACS analysis and sorting of frozen lymphocytes

frozen PBMCs were used for sorting dendritic cells and for surface staining. Cell sorting was performed by thawing 20×10⁶PBMCs at room temperature (RT) and washing them twice in medium at RT in 50-ml tubes in a 10-ml volume at 1,400 rpm. Cells were suspended to 20×10⁶/ml and stained with seven colors using Fc block, Lin-FITC, CD11c-APC, CD123-PE, CD3-Amcyan, CD4-Alexa700, CD25-Pacific Blue, and 7-AAD. Myeloid DCs were sorted on a FACS Aria (BD Biosciences), put in 350 μl of RLT (lysis buffer), and frozen at -70°C in Eppendorff microtubes.

For surface staining, freshly isolated PBMCs were suspended at 8–10×10⁶ cells/ml. Surface two to three color staining of cells were done with the following surface antibodies: CD4-FITC/CD25-PE, CD8-FITC/CD25-PE, CD3-APC/CD69-PE,CD11c-APC/Lin-FITC/CD86-PE, CD11c-APC/Lin-FITC/CD83-PE,CD11c-APC/Lin-FITC/HLAdr-PE. For LAP staining cells were preincubated with rLAP/control antibody for 20 min, and stained with CD4-FITC and CD25-PE or CD8-FITC. For Foxp3 surface staining, surface two-color staining of cells using CD4-FITC and CD25-PE was performed, followed by intracellular staining with IC/Foxp3-Alexa 647-APC or with CTLA4-APC.

5. Expression of cytokines by dendritic cells

total RNA was isolated from frozen sorted DC pellets using the RNA easy Mini Kit (Qiagen). RNAwas stored at −80°C. First-strand cDNA synthesis was performed for each RNA sample from 0.5–1 μg of total RNA using Taqman reverse transcription reagents (Applied Biosystems). cDNA was amplified using sequence-specific primers (for IL-23, IL-10, IL-6, and TGF-β) and real-time PCR mix (Applied Biosystems) on an ABI7500 cycler. The GAPDH gene was used as an endogenous control to normalize for differences in the amount of total RNA in each sample. All values were expressed as fold-increase or -decrease relative to the expression of GAPDH.

6. Antigen arrays

a panel of self and non-self proteins, peptides and lipids were spotted onto Epoxi slides (TeleChem, CA, USA) as described [15], a complete list of the antigens included in the arrays is provided in supplementary Table 1. Since several of the self-antigens included in our antigen microarrays are targeted by natural antibodies [16, 17], we could detect both the up- and down-regulation of preexisting IgG and IgM reactivities. Sera from OKT3-treated subjects were assayed at a 1/10 dilution and the IgG or IgM reactivities displaying significant changes upon treatment were identified. Briefly, antigens were spotted in replicates of six, the microarrays were blocked for 1 h at 37°C with 1% bovine serum albumin, and incubated for 2 h at 37°C with a 1:200 dilution of the test serum in blocking buffer. The arrays were then washed and incubated for 45 min at 37°C with a 1:500 dilution of detection antibodies: a goat anti-mouse IgG Cy3-conjugated antibody or a goat anti-mouse IgM Cy5-conjugated antibody (Jackson ImmunoResearch, West Grove, PA). The arrays were scanned with a ScanArray 4000X scanner (GSI Luminomics, Billerica, Massachusetts, USA) and the IgM and IgG results were recorded separately.

To compare the results from different arrays the raw data were normalized [15] and analyzed using the GeneSpring software (Silicon Genetics, Redwood City, CA). Antigen reactivity was defined by the mean intensity of binding to the replicates of that antigen on the microarray. To identify antibody reactivities that were consistently up- or down-regulated following treatment with OKT3 the data were analyzed with the Wilcoxon rank-sum test, a non-parametric test robust to outliers. The multidimensional nature of the data produced by the antigen arrays results in an increased probability of picking up “false positives”, this is controlled by using a False Discovery Method [18]. We used the Benjamini and Hochberg false discovery method with a p value of 0.2 to determine significance [18]. To identify antibody reactivities that show a similar behavior in response to OKT3 administration we used a hierarchical clustering method. The hierarchical clustering we performed using a pairwise average linkage algorithm based on Pearson’s correlation as a distance measure [18].

Statistical analysis

Differences were analyzed using Student’s t test. When there were more than two groups used for comparison, differences were analyzed by the one-way analysis of variance test. p values<0.05 were considered to be significant.

Safety of oral OKT3 and GC

The treatment was well tolerated by all subjects and no systemic effects were observed at any doses including changes in vital signs (temperature, pulse, blood pressure), and liver, kidney or hematologic measures (complete blood counts including differential), during treatment or follow-up (30 days post treatment). The oral OKT3 did not cause any GI symptoms such as diarrhea constipation, or irritable bowel disease.

Effect of oral OKT3 on cell surface CD3, lymphocyte count, and proliferation

Unlike what has been reported for treatment with IV anti-CD3 (which is given at a dose of 5 mg per day for 5 days), we observed no decrease in the CD3+ lymphocyte count or modulation of CD3 from the T cell surface Fig. 3. In addition, no subject developed anti-OKT3 (HAMA) antibodies. As shown in Fig. 4, we observed an increase in proliferation to anti-CD3 stimulation on day 5 that reverted to baseline by day 10. Figure 4 shows the proliferative responses in the three subjects that received 1.0 mg OKT3 orally (p<0.001) for day 5 vs. 0, and p<0.0013 for day 10 vs. 5). No significant differences in proliferation were observed in subjects dosed with 0.2 mg or 5.0 mg of OKT3. No additional effects were observed in the GC-treated groups.

Despite the increase in proliferation on day 5, Th1 and Th17 cytokine secretion was reduced by 43% and 41%, respectively, in the supernatants on day 5 and remained reduced on day 10. As shown in Fig. 5, we observed decreases in IFN-γ (p<0.004 days 5 or 10 vs. 0) and IL-17 (p<0.0044 for days 5 or 10 vs. 0). We observed a trend for an increase of IL-13 and TGF-β on days 5 and 10 but these changes were not statistically significant. We observed no significant changes in proliferation or cytokine production in untreated subjects evaluated at the same schedule and no additional effects in GC-treated subjects. These results demonstrate that OKT3 is biologically active when given orally and decreases the pro-inflammatory profile as measured by IFN-γ and IL-17.

Oral OKT3 increases IL-10/TGF-β and decreases IL-23 and IL-6 expression in dendritic cells

To investigate the effect of oral OKT3 on the innate immune system, we measured the expression of IL-10, TGF-β, IL-23, and IL-6 in dendritic cells by rtPCR. As shown in Fig. 6 for the subjects dosed with 1.0 mg OKT3, we observed an increase in IL-10 and TGF-β (p<0.005 for days 5 or 10 vs. 0). An opposite pattern was observed for IL-23 and IL-6 where we observed a decrease in IL-23 expression (p=0.006) and IL-6 expression (p=0.005) for days 5 or 10 vs. 0. No significant changes were seen in the 0.2- and 5-mg dose groups. In the 1-mg OKT3+GC-treated group, we also found increased expression of IL-10 and TGF-β in DCs which was more apparent in day 5 than in subjects with OKT3 (p<0.005 for day 5 vs. 0; Fig. 7). This is consistent with reports that glycolipids affect the function of DCs [19-22]. We observed no significant changes in DC cytokine profiles in the untreated subjects evaluated at the same schedule. These results demonstrate that oral OKT3 affects the anti-inflammatory profile of DCs.

Effect of oral OKT3 on T cell regulatory markers

We measured immunophenotypic markers associated with regulatory T cells, including CD25hi, Foxp3 and CTLA4. We found a significant increase (p<0.005) in Foxp3 expression on CD25hi cells as measured by MFI (Fig. 8a) on day 5. Similarly, CD25hi CTLA4+ cells MFI increased on day 5 (Fig. 8b). We also found an increase in the expression of TGF-β on CD25int/lo T cells (Fig. 8c; p<0.005 for day 5 vs. 10) and an increase in CD8+CD25+cells (Fig. 8d). We were unable to perform functional studies due to limits in cell numbers. No significant changes in regulatory T cell markers were observed in the untreated subjects evaluated at the same schedule. In animals, induction of regulatory T cells is the primary mechanism by which oral anti-CD3 suppresses disease. Although our phenotype data suggests induction of regulatory T cells in humans, demonstrating that oral anti-CD3 induces functional regulatory T cells in humans will require further studies. Of note, we did not observe changes in the surface expression of CD40, CD80, CD83, CD86, HLA-DR, or LAP in subjects orally administered OKT3.

Effect of Oral OKT3 on Antigen Arrays

Antigen microarrays constitute a new tool for the study of the immune system in health [15, 23-25] and disease [26, 27]. We used an antigen microarray containing a broad panel of antigens (n=550) that included self and non-self proteins, heat shock proteins, and infectious agents to investigate the effects of oral OKT3 on the immune repertoire. We measured both increases and decreases of immunoglobulin reactivities.

Figure 9a shows that treatment with OKT3 resulted in a dose-dependent change in the T-cell-dependent IgG repertoire. Minimal changes were observed at the 0.2-mg dose (only four reactivities affected) whereas at 1.0 mg, 37 IgG reactivities were affected and at 5.0 mg, 65 IgG reactivates were affected (Fig. 9a). At the 1.0 mg dose there was an equal number of down-regulated (n=19) as up-regulated (n=18) reactivities, whereas at the 5-mg dose, more up-regulated reactivates were observed (47 vs. 18). Figure 9b shows a heatmap of changes in the IgG repertoire following oral administration of 1.0 mg OKT3 in the three individual subjects. No changes occurred in the IgM repertoire. The changes we observed affected a range of reactivities. We did not perform antigen assays in subjects that received OKT3 plus GC.