Overlapping molecular signaling of IRX-2 and Ta1 resulting in synergistic biological activity

1. Introduction

With the recent approvals of immune therapies such as checkpoint inhibitors and chimeric antigen receptor cell therapy, the development of immune modulators for cancer therapy has been given new life. Thus, the vision of John Hadden that immunopharmacolgy would be a key component of cancer therapy is coming to fruition. Two of the pioneering agents in immunopharmacology are thymosin a1 and IRX-2, and their synergy in combination is the subject of the studies reported in this article.

Thymosin a1 (a 28aa peptide) was isolated from a lymnphopoetic thymus extract (thymosin fraction 5) and its initial demonstration of bioactivity was in a thymocyte differentiation assay (i.e. the rosette assay) [1,2]. Subsequent studies by many investigators demonstrated that Ta1 was a pleotropic peptide with multiple activities as presented in Table 1 [3–11].

Table 1. Complementary and similar activities of IRX-2 and Tα1*.

IRX-2	Thymosin a1
Antigen presenting cells
Restores DC function in cancer patients	Increases differentiation and activation
Upregulates APC machinery in cancer patients	Activation of CD14^+
Increases T-cell stimulatory capacity
T cells	Increases T cell activation marker expression
Increases proliferation
Corrects anti-apoptotic balance in cancer patients	Inhibition of T cell apoptosis
Stimulates Jak3, Stat 5, Akt, PI3K anti-apoptosis pathway	Stimulates through MyD88/NfkB+ pathway
Anti-tumor activity
Activity in B16 model	Activity in B16 model
Lewis Lung carcinoma
SSCHN1	Friend erythroleukemia
Increases T-cell responses to protein, peptide, cellular, and viral cancer vaccines	Colorectal cancer
Natural killer cells and other cell types
Restores NK cell activity from cancer patients	Increases NK cell function
Increases ADCC	Enhances T cell dependent antibody responses to vaccines
Reverses RT or HC-induced leukopenia	Reverses leukopenia to multiple stimuli
Stimulates HPV-suppressed Langerhan’s cells	Increases gene expression of multiple immune-related genes

*Summary of observations from Refs [1–25].

IRX-2 is a combination cytokine biologic with multiple activities as a result of the individual and combined activities of its constituents (Table 1) [12–18]. IRX-2 is currently being evaluated as an adjuvant therapy for patients with head and neck cancer in a randomized placebo-controlled phase 2b study. Given for 10 days before the surgery, IRX-2 appears to enhance lymphocyte infiltration of the tumor and to prolong survival as compared to historical controls. Although the majority of patients benefit as defined by enhanced survival due to lymphocyte infiltration, a number of patients failed to benefit. Thus, the combination of IRX-2 with another biologic such as Ta1 is an attractive hypothesis with respect to generating lymphoid cells which may be missing in those individuals who fail to respond to IRX-2 [19–25].

2. Studies demonstrating effectiveness of Ta1 plus IRX-2

2.1. Preclinical studies

Early studies by the Hadden group, with the combination of Ta1 and IRX-2, were performed in hydrocortisone-suppressed mice and the combination was shown to be superior to either agent alone [19]. Based on those studies and the observation by the Garaci group that Ta1 and IL-2 were superior to either alone, studies were undertaken to combine Ta1 with IRX-2 [20,21,23,24]. The studies suggested synergy, and as shown in Figure 1, significant decrease in tumor growth and survival can be demonstrated in the LLC model.

Figure 1. Tumor size in C57Bl/6 mice by days post inoculation. There were 10 mice per treatment group and the size of the tumor (y axis cm²) for the day of measurement (x-axis) is indicated in the graphs. Mice were randomized on day 10 following inoculation with Lewis Lung Carcinoma cells to assure similar tumor size and a non treatment group is shown on the upper left (A). The remaining three groups received cyclophosphamide (cyclo) on day 11. Three days after cyclo (day 14), Ta1 was administered to one group (lower right D) for three consecutive days. IRX-2 was given beginning on day 17 for 30 days (lower right (C) and left (D)). Survival is also indicated above the graphs for each group at day 62 (end of measurements) and day 92. Mice receiving CY and Ta1 alone were not different from the CY only control. Data replotted from published data in reference 24.

2.2. Clinical studies

Patients who have received surgery and radiotherapy for their tumors are often immune-suppressed. In a phase 2 clinical study with these immune suppressed patients, they were treated with Ta1, IRX-2, or the combination. [22,25] Immune suppression was defined as lower than normal CD3 cells in their blood (Table 2) and treatment was for 10 days with Ta1, IRX-2, or two separate but same-day injections of Ta1 and IRX-2. Ta1 had no effect on CD3 cells, as compared to IRX-2 and the combination. As shown in Figure 2, increase in T cells with the combination treatment was due to an increase in CD8 cells as compared to CD4. The increase in CD8 cells would be predicted to be an important component of an antitumor response. In support of this immune enhancement hypothesis, clinical improvements in the 4 Ta1+IRX-2 which were not seen in the IRX-2-only group included clearance of venereal warts, oral candidiasis, papilloma virus, and/or Candidiasis albicans.

Table 2. T-cell-deficient patients and treatment to reverse deficiency [22,23].

Treatment*	Ta1***	IRX-2****	Ta1+IRX-2*****
Number of patients**	4	7	7
Frequency	10 daily injections	10 daily injections	10 daily injections
Average CD3 (400–1600 cells/ul for normal patients	395	455	336

*Injected sc to target cervical lymph nodes.

**HNSCC patients with lymphopenia due to RT but free from cancer.

***Ta1 licensed in Mexico and purchased for study.

****IRX-2 prepared at INCAN under similar process as for cGMP Studies in US.

*****Administered separately but on the same days.

Figure 2. Effect of 10 daily treatments with IRX-2 or IRX-2+ Tα1 protocol on total lymphocyte count, mature CD3 + T lymphocyte, CD8+, CD4 + T cells in blood of seven patients with lymphocytopenia following radiation treatment. Data are expressed as the delta (increase) from day 0 to day 11 in cell/L plus/minus SEM. Replotted from Reference 25. Additional minor lymphoid cells in the IRX-2+Ta1 patients were identified as B cells (CD19), monocytes (CD16), NK/NKT (CD56 CD57) plus cells with neither T or B cell markers (‘null cells”) .

3. Discussion

Our hypothesis is that the preclinical and clinical studies suggest that in settings of immune deficiency (due to either the tumor or chemo/radiation therapy), Ta1 acts to generate cells which are capable of responding to the cytokines in IRX-2 and therefore generates a more effective immune response than either alone. It is especially attractive to speculate that the patients in the IRX-2 clinical trial who failed to respond may have benefited from pretreatment with three days of Ta1 prior to IRX-2. The ongoing randomized phase 2 trial may result in the identification of pre surgery biopsy profiles which predict a failure to response to IRX-2 and these patients may be a candidate for the combination therapy. Alternatively, in patients who are scheduled to receive boosters of IRX-2, having an arm for patients who receive three days of Ta1 prior to IRX-2 may be a valuable option for evaluating the effectiveness of combination therapy.

4. Expert opinion

Combination biologic therapies are important due to the observation that most immune therapies are not successful in all patients. The use of combinations is thought to be a way to address this problem. While natural combination biologics such as IRX-2 were thought to resolve this issue, there are still patients that failed to respond to IRX-2. This editorial reviewed the data which suggests that adding another pleotropic biologic (thymosin alpha1) will enhance the activity of both biologics, especially as related to tumor models and immune-deficient patients. The biggest challenge in the field is that clinical studies are required to confirm the data presented in this review. Given that immunotherapy is becoming a well-funded and promising are, it is possible that future clinical trials with the combination of Ta1 and IRX-2 may be initiated. The experiments discussed in this review also provide support for the possibility that these two biologicals might be combined either separately or in combination with other immunotherapies such as the CTLA-4/PD-1 reagents.

Declaration of Interest

P. Naylor has served as a consultant to IRX Therapeutics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper has been published as part of a supplement issue covering the proceedings of the Fifth International Symposium on Thymosins in Health and Disease and is funded by SciClone Pharmaceuticals.