OPRX-106 consists of lyophilized Nicotiana tabacum (BY2) tobacco plant cells expressing the recombinant TNFR2-Fc fusion protein (rTNFR2-Fc), cultivated in a bioreactor system ProCellEx. The rTNFR2-Fc consists of the soluble form of the human TNF2 receptor fused to the Fc fragment of a human IgG1 antibody domain which imparts it a longer serum half-life. Plant cell wall which contains cellulose, serves as a natural protective agent against the gastric environment. The amino acid sequence of rTNFR-Fc is similar to the sequence of the approved anti-TNFR agent etanercept.1,2
OPRX-106 has been evaluated to be an effective anti-tumor necrosis factor alpha (TNFα) therapy. It is also being explored as a means for exerting a beneficial immune response through local biological effects in the gut, with no systemic absorption and with better safety relative to currently approved anti-TNFα proteins.
Oral immune therapy is based on the concept of oral administration of nonabsorbable compounds which target the gut immune system to redirect the systemic immune system toward an anti-inflammatory direction, without immunosuppression.3,4
Preclinical studies showed that oral administration of OPRX-106 alleviated immune-mediated liver injury in a concanavalin model. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were decreased and were comparable with those in mice which had received high-dose steroids. The beneficial effect was also observed as a marked decrease in hepatic necrosis.1 1 2
The safety and the exploratory immune modulatory effects of orally administered OPRX-106 were shown in a phase I study in humans. Three different doses (2, 8, or 16 mg/d) of OPRX-106 were orally administered for 5 consecutive days in 14 healthy volunteers. Treatment was found to be safe and well tolerated. The pharmacokinetic (PK) study showed that OPRX-106 is not absorbed into circulation. No effect on white blood cells (WBC) and lymphocyte counts was noted. A dose-dependent effect was observed on systemic lymphocytes. The oral administration of all 3 dosages was associated with an increase in CD4+CD25+ and CD8+CD25+ subset of suppressor lymphocytes. An increase in CD4+CD25+FoxP3 Tregs was noted in the group treated with 8 mg. Also, NKT regulatory cells, CD3+CD69+, and CD4+CD62 lymphocyte subsets increased with treatment . No change in serum TNFα was observed.5
The present study was aimed at determining the safety PK and exploratory efficacy of orally administered human TNFR fusion protein expressed in plant cells, in patients with mild to moderate ulcerative colitis (UC).
METHODS
Study Design and Product
The clinical trial was a phase 2a, open-label, randomized study, which examined the safety, PK, and exploratory efficacy of OPRX-106 in patients with UC. The subjects were enrolled in 8 centers in Israel, Serbia, and Bulgaria. OPRX-106 consisted of lyophilized, genetically modified plant cells expressing the human tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc), a dimeric soluble fusion protein of the TNF-2 receptor protein and the Fc fragment of IgG1. The product was manufactured by the sponsor Protalix Ltd. (Carmiel, Israel). The trial was registered at ClinicalTrials.gov, NCT02768974.
Study Population
Eligible subjects were adult males or females who were 18 to 70 years of age. Patients had to be diagnosed with UC according to the following European guidelines for a minimum of 3 months: medical history, physical examination, laboratory tests [anemia, increased levels of C-reactive protein (CRP), fecal samples for blood], historical histopathological evidence from flexible sigmoidoscopy or colonoscopy showing active mild-to-moderate UC, and a high level of calprotectin in stool (>100 μg/mg). Exclusion criteria included a history of colonic or rectal surgery other than hemorrhoidal surgery or appendectomy, patients receiving total parenteral nutrition or severe UC evidenced by the following signs of toxicity: heart rate >90 beats/min at rest and a temperature >38.0°C. Other exclusion criteria included ulcerative proctitis with disease limited to <15 cm from the anal verge, use of >4.8 g of 5-aminosalicylic acid (5-ASA) or equivalent, corticosteroid or 5-ASA enemas, foams, or suppositories within 2 weeks before screening or at any time during the study, use of anti-inflammatory drugs (cromones, xanthines, leukotriene antagonists) or natural remedies (probiotics, omega-3 fatty acids) within 4 weeks before screening or at any time during the study, use of oral or parenteral antibiotics within 2 weeks before the screening or at any time during the study, use of chronic nonsteroidal anti-inflammatory (NSAID) therapy, use of immune suppressive agents including anti-TNF agents, azathioprine, mercaptopurine (6MP), or methotrexate 12 weeks before screening or at any time during the study and use of steroids 12 weeks before screening or at any time during the study.
Study Design
Twenty-five UC patients were enrolled for this study. However, 1 subject withdrew consent before study drug administration. Therefore, 24 UC patients of 18 years and above were finally enrolled in the study including 11 males and 13 females with a mean age of 42.63±13.74 years (range, 23 to 73 y), who received 2 or 8 mg of OPRX-106 at least once daily, for 8 weeks. On day 1 and on week 8, the subjects were monitored for 6 hours succeeding OPRX-106 administration for PK sampling. Physical examination and blood samples for testing liver and kidney functions and complete blood count (CBC) were performed at screening and on weeks 1, 2, 4, 6, 8, and 10 (follow-up visit). A follow-up lower endoscopy was performed on week 8. Adverse events (AEs) and concomitant medications were evaluated at each study visit.
Pharmacokinetics Study
Blood samples were tested for the presence of OPRX-106 levels at baseline visit and on week 8 before study drug administration and at 1, 2, 4, and 6 hours after study drug administration.
Flow Cytometry Analysis of Peripheral Blood Lymphocytes
The immune modulatory effects of OPRX-106 on lymphocyte subsets were evaluated by flow cytometry analysis on day 1, week 4, and week 8. The following protocol was followed for the flow cytometry analysis. Cells were suspended in 100 μL fluorescence-activated cell sorting (FACS) buffer (2% bovine serum albumin, in phosphate-buffered saline) and incubated with the following surface antihuman antibodies: antihuman CD4-APC, antihuman CD8-Pacific Blue (PB), antihuman CD16-APC-Alexa Fluor 700 (APC-AF700), antihuman CD25-FITC, antihuman CD45-Krome Orange (KO), antihuman CD56-APC-Alexa Fluor 750 (APC-AF700), and antihuman CD62L-FITC (Beckman Coulter) for 15 minutes. Tubes stained for intracellular staining with Foxp3 antibody (anti-FoxP3-PE, Beckman Coulter) were fixed and permeabilized followed by incubation for 1 hour. Cells were washed with FACS buffer and analyzed. Cell phenotyping was performed by Navios 10 colors 3 Lasers (Beckman Coulter) and analyzed by Kaluza software. The FACS analysis was performed at AML-American Medical Laboratories, Tel Aviv, Israel. Only the live cells were counted, and background fluorescence from non–antibody-treated lymphocytes and isotype control was subtracted.
Serum Cytokines
To determine the potential effects of orally administrated OPRX-106 on cytokine levels, serum levels of TNFα, interferon gamma (IFN-γ), IL-6, IL-10, and IL-12 (p70) were determined by highly sensitive enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s instructions. TNFα, IL-6, IL-10, and IL-12 (p70) were quantified by ELISA using Quantikine (R&D Systems, Catalogue number: HSTA00E, HS600B, HS120, and HS120, respectively), and IFN-γ was quantified using HS (eBioscience, Catalogue number: BMS228HS). The analysis was performed at American Medical Laboratories (AML), Herzliya Pituach, Israel.
Efficacy and Safety Assessments
Study endpoints included safety, PK, and exploratory efficacy parameters. Clinical response (improvement) at baseline versus week 8 was defined by the following criteria: decrease in the Mayo score of at least 3 points, decrease in the subscore for rectal bleeding of at least 1 point, a rectal bleeding subscore of 0 or 1, clinical remission at week 8 which is defined as clinically symptom-free, Mayo Score ≤2 with no individual subscore exceeding 1 point after treatment , histopathological improvement in Geboes histologic grading from baseline to week 8, improvement in high sensitivity C-reactive protein (hs-CRP) levels from baseline to week 8, improvement in fecal calprotectin levels from baseline to week 8, and changes in systemic immune modulation parameters from baseline to week 8. Mayo responsiveness and remission were defined as a decrease in the total Mayo score; reductions in Geboes grading, CRP, and calprotectin were exploratory secondary endpoints improvements of any degree between baseline and week 8 were calculated as response. Endoscopic scoring was performed at each site by the investigator at the start and end of the study. Mucosal healing was an exploratory secondary endpoint and any improvement from baseline to week 8 was considered a response. Histology was evaluated by a central reader blinded pathologist. AEs were evaluated at each study visit. These were defined as any unfavorable and unintended signs, symptoms or disease that seemed during the study period whether considered related to the study drug, including accidental injuries, reasons for any change in medications, reasons for admission to hospital or reasons for surgical procedures and any laboratory abnormality assessed as clinically significant by the investigator. The AEs were monitored from the start of the treatment until 2 weeks after the final visit dose.
Statistical Analysis
Quantitative data were summarized with mean and standard error, SD, median, minimum, and maximum values for both actual and change from baseline results. Qualitative data were summarized to show frequency and percentage within each category. Data manipulation, tabulation of descriptive statistics, calculation of inferential statistics, and graphical representations were performed using SAS (v9.3 or higher) for Windows.
RESULTS
Patients
Twenty-five patients with mild-to-moderate UC were enrolled in the trial and 24 received the study drug (1 patient withdrew consent before receiving the first dose of the study drug). Out of 24 patients, 22 (92%) were classified as having moderate UC based on a Mayo score >6. Table 1 summarizes the patient characteristics in each of the 2 groups.
TABLE 1 -
Patients Characteristics
Parameter
2 mg/d (n=13)
8 mg/d (n=11)
Mean age (y)±SD (range)
42.62±10.41 (28-63)
42.64±17.43 (23-73)
Male:Female
6:7
5:6
Ethnicity
Caucasian
13
11
Mean baseline values (±SD)
2 mg/d
8 mg/d
Mayo score*
7.69±1.11
6.82±1.83
Mayo Endoscopic subscore
2.2±0.4
2.0±0.9
Geboes score
12.00±4.76
11.00±6.60
Two subjects discontinued due to UC exacerbation, 1 subject discontinued due to lack of response, 1 subject discontinued due to usage of antibiotics treatment .
*Two subjects discontinued due to lack of response.
UC indicates ulcerative colitis.
Pharmacokinetics
The variable OPRX-106 concentrations Cmax and Tmax indicated no or minimal absorption of OPRX-106 into the blood circulation. Out of 24 subjects, 9 had no detectable OPRX-106 level at any point in time, 15 subjects had at least 1 sample with detectable but insignificant levels of OPRX-106 ranging from 46 to 112 pg/mL.
Antidrug Antibodies
A total of 80 serum samples from 24 subjects were tested (in duplicates) for the presence of anti-OPRX-106 antibodies with no evidence for anti-OPRX-106 antibodies being observed.
Safety
Oral PRX-106 was well tolerated and no serious AEs were reported. None of the patients discontinued the therapy due to AEs. The dropout rate was consistent with other trials in similar populations. A total of 40 AEs were reported in 15 (63%) patients with 95% (38) of the AEs being mild to moderate and 5% (2) being severe AEs (nausea defined as possibly related). Of these events, 40% (16/40) were reported as treatment -related. Other events recorded were headache (4), increased creatine phosphokinase (CPK) (2) and 1 complaint of each of the following events: dysphagia, nausea, chills, fatigue, peripheral edema, increased appetite, dizziness, pruritus, hypertension, and eosinophilia. 60% (24/40) of AEs were reported as not related. No difference was noted between doses (2 or 8 mg). Table 2 summarizes the AEs.
MedDRA System Class
Preferred Term
N (Events)
Blood and lymphatic system disorders
Anemia
2
Eosinophilia
1
Hypochromic anemia
1
Eye disorders
Dry eye
1
Gastrointestinal disorders
Abdominal pain
1
Anal fissure
1
Colitis ulcerative
7
Constipation
1
Diarrhea
1
Dysphagia
2
Hemorrhoids
1
Nausea
1
Vomiting
1
General disorders and administration site conditions
Chills
1
Fatigue
1
Influenza like illness
1
Edema peripheral
1
Pyrexia
1
Infections and infestations
Nasopharyngitis
1
Pharyngitis
1
Upper respiratory tract infection
1
Investigations
Blood creatine phosphokinase increased
2
Metabolism and nutrition disorders
Increased appetite
1
Nervous system disorders
Dizziness
1
Headache
4
Psychiatric disorders
Sleep disorder
1
Skin and subcutaneous tissue disorders
Pruritus
1
Vascular disorders
Hypertension
1
Figure 1 shows the beneficial effect of OPRX-106 as seen by an improvement in the Mayo score. A total of 12 patients (67%) achieved a clinical response, and 5 achieved clinical remission (28%). No significant differences were noted between the 2 dosage groups.
FIGURE 1: Following 8 weeks of treatment with OPRX-106, 28% of the patients achieved clinical remission and 67% of the patients achieved clinical response; 89% of the patients had any improvement in their Mayo score. 33% of the patients achieved mucosal healing and 61% of the patients had mucosal improvement.
Figure 1 shows the beneficial effect of OPRX-106 as seen by an improvement in mucosal healing. A total of 11 patients (61%) showed improvement in their endoscopy score, defined as a decrease in endoscopy subscore at week 8. Six patients achieved mucosal healing (33%). No significant differences were noted between the 2 dosage groups.
Improved Geboes score was noted in 11/18 (61%) patients. A reduction in calprotectin levels was demonstrated in 13/18 (72%) patients, and a reduction in hs-CRP serum levels were observed in 14/18 (78%) patients, further supporting a systemic anti-inflammatory effect of the drug.
Table 3 shows that the response to therapy was associated with a trend for a decrease in IFNγ, TNFα, and interleukin 6 (IL-6) serum levels of pro-inflammatory cytokines. Panel A shows the percentage of patients with decreased IL-6 levels arranged according to decreasing Geboes score. Panel B shows the percentage of patients with reduced IFNγ and IL-6 classified by clinical remission and response. The data show a trend for a correlation between the clinical effect of OPRX-106 and the systemic anti-inflammatory effect. No relationship was seen between the clinical effect and the effect on reduction of serum TNFα levels.
TABLE 3 -
Effect of OPRX-106 on Cytokine Levels
No. Patients With Decreased Levels of Cytokine at Week 8 Compared With Baseline
Clinical Response or Remission (Based on Mayo Score)
Cytokine
All (N=18)
Yes (n=12)
No (n=6)
IL-6
7
6
1
TNFα
11
7
4
IFNγ
10
7
3
Decreased Geboes score
All (N=18)
Yes (n=11)
No (n=7)
IL-6
7
5
2
TNFα
11
6
5
IFNγ
10
6
4
IFNγ indicates interferon gamma; IL-6, interleukin 6; TNFα, tumor necrosis factor alpha.
A similar trend was demonstrated with an increase in the CD3+CD4+CD25+Foxp3+ Tregs in patients that achieved clinical response/remission (Table 4 ). Taken together, the effect on the systemic immune system was correlated with the clinical and laboratory response to orally administered nonabsorbable OPRX-106.
TABLE 4 -
Effect of OPRX-106 of Regulatory T Cells
No. Patients With Increased CD3+ CD4+ CD25+ FoxP3+Population at Week 8 Compared With Baseline
Achieving Clinical Response or Remission (n=11)
Others* (n=6)
6
3
*Patients who did not achieve clinical response/remission in the study.
DISCUSSION
The results of the study show that oral administration of OPRX-106 was safe, well tolerated and clinically effective in patients with mild-to-moderate UC. OPRX-106 was minimally absorbed systemically as shown in the PK analysis, and from the lack of antidrug antibodies in all treated patients. OPRX-106 had no major side effects. No immune suppression was noted as shown by lack of bone marrow suppression or alterations in subsets of lymphocytes. Although not being systemically absorbed and suppressing the immune system, OPRX-106 further exerted a beneficial anti-inflammatory effect on the systemic immune system. This was manifested by a decrease in serum levels of the pro-inflammatory cytokines IFNγ and IL-6, which correlated with the clinical response. Similarly, an increase in the CD3+CD4+CD25+Foxp3+ subset of lymphocytes correlated with clinical response as well. The beneficial effect on the systemic immune system had a favorable clinical impact in disease parameters. The data show that 67% patients achieved clinical improvement of which 28% achieved clinical remission. Reduction in calprotectin and hs-CRP levels and improved Geboes score were noted in majority of the tested patients.
Biological agents that target TNFα improved the therapeutic approach to inflammatory diseases.6,7 8 8–10 treatment failures.8,11 treatment .12,13
Etanercept is a recombinant, dimeric, soluble TNFR fusion protein that blocks only soluble TNF but not the membrane‐bound TNF.14 6,15 treatment of Crohn’s disease (CD) or UC.14,16 17 treatment suggests differences between etanercept and other anti-TNFα antibodies in molecular structures, TNF neutralizing effects and PK.18–20 21 22 23 24 25 24
Loss of response (LOR) to anti-TNF therapy is common among patients with IBD and presents a challenge to physicians in the management of symptoms.26 27,28 29 30 27 31 32 33 34
The oral administration of nonabsorbable OPRX-106 may overcome the LOR in patients with IBD better than the parenteral administration of etanercept as well other anti-TNF-based formulations, reflecting its potential to induce a systemic beneficial immune modulatory effect by targeting the gut immune system. Oral administration of OPRX-106 also overcomes some of the side effects encountered by parenteral administration, including the long-term effects of immune suppression. OPRX-106 might exert a local impact on the gut-associated lymphoid tissue, which does not exist when parentally administered. As this drug is not absorbed when orally administered, postabsorption signaling from the gut exerts an additive or synergistic effect on the gut-associated lymphoid tissue.4 35–40 41,42 43,44 4,45–47 1 45,48
The oral administration of OPRX-106 promoted CD4+CD25+FoxP3+ Tregs, thereby reducing the serum levels of pro-inflammatory cytokines. Dysregulation of Tregs is a significant risk factor in conferring human autoimmune diseases.49 50 51,52 53 54 55–58
In conclusion, oral administration of plant cells expressing recombinant anti-TNF fusion protein shows a beneficial biological activity in UC patients. An orally delivered nonabsorbable OPRX-106 was safe, well tolerated, and did not have any side effects associated with general immune suppression. The oral delivery of therapeutic proteins has been a long-term goal with relatively limited success. Lack of systemic absorption, absence of a change in systemic WBC, and the systemic immune modulation documented by alteration of different subsets of lymphocytes, support the notion that oral OPRX-106 may overcome some of the obstacles encountered by parenteral administration of currently used anti-TNF agents. It may provide an answer to the long-term immune suppression encountered in patients with chronic disorders who use these agents for prolonged periods of time, in addition to LOR due to neutralizing antibodies. OPRX-106 may provide oral, safe, and effective anti-TNFα-based therapy for IBD. Although the current study was an open-labeled uncontrolled study, the data support the further development of OPRX-106 in controlled studies in patients with IBD and other immune-mediated disorders.
ACKNOWLEDGMENTS
The authors thank the investigators and their patients for their participation in the study.
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