Interleukin 2 (IL-2), discovered in 1976, is a type of T cell growth factor (TGGF) that can support the long-term growth of T cells in vitro. IL-2 is primarily secreted by CD4+ T cells after activation through the T cell receptor (TCR) and CD28 co-stimulatory signals. CD8+ T cells, NK cells, natural killer T cells (NKT), and dendritic cells also produce IL-2, but in smaller quantities.

IL-2 exerts its effects through the IL-2 receptors (IL-2Rs) on the cell surface. The IL-2R is composed of three subunits: IL-2Rα (encoded by IL2RA, also known as CD25), IL-2Rβ (encoded by IL2RB, also known as CD122), and IL-2Rγ (encoded by IL2RG, also known as CD132). These subunits can form multiple combinations, with the functional forms being the intermediate affinity dimer (IL-2Rβ-IL-2Rγ) and the high affinity trimer (IL-2Rα-IL-2Rβ-IL-2Rγ). The former is constitutively expressed mainly on memory CD8+ T cells and NK cells, while the latter is constitutively expressed primarily on regulatory T (Treg) cells, indicating that Treg cells are more sensitive to low levels of IL-2 compared to other cell types.

Figure 1. Diagram illustrating the binding of IL-2 to low affinity, intermediate affinity, and high affinity IL-2 receptors [1]

IL-2 for Treatment of Diseases

As research has progressed, it has been discovered that IL-2 has dual activities. High doses of IL-2 can promote the proliferation of effector T cells, thereby exerting anti-cancer effects, while low doses of IL-2 can selectively activate Treg cells, thus inhibiting immune activation. Therefore, IL-2 can be applied in both the field of anti-tumor therapy and the field of autoimmune disease treatment.

IL-2 & Cancer Treatment

IL-2 is the first cytokine approved by the FDA for tumor immunotherapy. It can activate NK cells, cytotoxic CD8+ T (CTL) cells, and lymphokine-activated killer (LAK) cells. Additionally, it can induce other immune cells to produce cytokines such as IFN, TNF, and CSF, which synergistically enhance the activity of immune cells.

Research shows that IL-2 and lectin-activated CTLs can effectively destroy tumor cells in vitro, while NK cells can directly kill tumor cells in the bloodstream and tumor tissues. Furthermore, IL-2 can induce the production of LAK cells, which specifically kill tumor cells. The surface of cancer cell membranes contains an antigenic determinant that can be recognized by LAK cells, whereas normal cells lack this determinant, allowing LAK cells to selectively target and kill tumor cells.

IL-2 and Autoimmune Diseases

Autoimmune diseases are characterized by reduced IL-2 secretion and dysregulated immune cell responses, leading to Treg cell dysfunction and excessive proliferation of effector cells, particularly CTL cells and CD4+ T cells. Treg cell dysfunction has been observed in many inflammatory/autoimmune diseases, such as type 1 diabetes (T1D) and systemic lupus erythematosus. Since IL-2 can expand Treg cells and restore CD25 expression, the application of exogenous IL-2 represents a potential therapeutic option for various autoimmune diseases.

Current Status of IL-2 Drug Development

As of today, the FDA has approved four IL-2 drugs, all of which are recombinant IL-2. Aldesleukin (Proleukin®) became the first US Food and Drug Administration approved immunotherapy for the treatment of metastatic renal cell carcinoma (RCC) and metastatic melanoma in 1992 and 1998, respectively. However, due to its short half-life, narrow therapeutic window, significant toxic side effects, and concurrent stimulation of Treg activation, its clinical use is limited.

Below list the approved IL-2 drugs:

• • Proleukin (aldesleukin) / Merck (MSD), Novartis, Iovance Biotherap

• • Simulect (basiliximab) / Novartis

• • Zinbryta (daclizumab) / Biogen, AbbVie

• • Ontak (denileukin diftitox) / Eisai, TSD Japan

Development of Engineered IL-2

In recent years, with a deeper understanding of the biological mechanisms of IL-2 and advancements in engineering technologies, the development of new IL-2 drugs has been active. Efforts have been made in various directions to optimize IL-2, including polyethylene glycol modification (PEGylation) of IL-2 molecules and fusion proteins (such as HSA, antibody Fc, and antibody fusions).

PEGylated IL-2 Agonist

Protein PEGylation is the most commonly used method to extend the half-life of protein drugs in vivo, while also increasing protein hydrophilicity and reducing immunogenicity. PEGylation at specific sites of IL-2 can also alter its receptor bias. By PEGylation at specific sites, IL-2 tends to bind IL-2Rβγc, leading to more specific activation of Teff in the tumor microenvironment, reducing its ability to activate Treg cells. This can maximize the anti-tumor effects of IL-2 and reduce its side effects such as capillary leakage.
Huateng Pharma has been deeply involved in the field of  PEG derivatives for over 20 years, specializing in providing high-quality PEG derivative products. We offer comprehensive support to meet your PEGylation needs, from laboratory research to scaled-up production levels!

NKTR-358: Fails in Phase II

NKTR-358 was the second novel IL-2 conjugate designed by Nektar using polymer conjugation technology. While traditional IL-2 has a half-life of only 1-2 hours, NKTR-358 significantly extends this half-life, requiring only 1-2 injections per month. What sets NKTR-358 apart from other immunotherapy drugs is its ability to selectively promote the proliferation and activation of Tregs in the body, addressing potential immune system imbalance issues in patients with autoimmune diseases. However, despite promising prospects, NKTR-358 failed in Phase II trials.

Figure 2. NKTR-358

The ISLAND study (NCT04433585) enrolled a total of 291 patients with moderate to severe SLE, aiming to evaluate the therapeutic effects of low dose (300µg, Q2W), medium dose (900µg, Q2W), and high dose (1800µg, Q2W) of NKTR-358 in SLE patients. The primary endpoint of the study was a 4-point reduction in the SLEDAI-2K score in pre-defined study populations. 

The results showed that compared to the placebo group, there was no significant difference in the proportion of patients with a reduction of ≥4 points in SLEDAI-2K score in any of the three dose groups. Among the three dose groups, the improvement in the medium dose group was the most significant, but it still did not reach a significant level. Meanwhile, none of the three dose groups achieved a significant improvement in the key secondary endpoint of British Isles Lupus Assessment Group (BILAG)-Based Composite Lupus Assessment (BICLA) response. Additionally, biomarker data indicated that NKTR-358 could promote dose-dependent proliferation of Treg cells, consistent with previous studies.

Figure 3. ISLAND study Primary endpoint [2]

Lilly once had high hopes for NKTR-358. In July 2017, Lilly acquired global rights to NKTR-358 for a total transaction value of $400 million, while the Phase I clinical data for NKTR-358 had not yet been disclosed. Additionally, Lilly was willing to share 75% of the costs for the Phase II development of the product for SLE and committed to bearing all its commercialization costs.

However, things didn't go as planned. Lilly had to abandon advancing NKTR-358 for the treatment of SLE in Phase III clinical development and pinned hopes on its upcoming Phase IIb trial for atopic dermatitis. Additionally, both parties agreed to collaborate on clinical studies for the treatment of psoriasis with NKTR-358.

Figure 4. NKTR-358 clinical trials

NKTR-214: Ultimately Abandoned

NKTR-214 is the first IL-2 drug developed by Nektar, an engineered immunostimulatory IL-2 cytokine prodrug composed of IL-2 molecules conjugated with an average of six PEG chains. It aims to exert anti-tumor effects by binding to IL-2Rβγ receptors associated with the activation and expansion of CD8+ T cells and NK cells without binding to the regulatory T cell IL-2Rαβγ receptors that suppress the immune system. Additionally, NKTR-214 is designed for sustained activation of the IL-2 pathway by slow release through its PEG chains, extending the half-life of IL-2 to approximately 20 hours, thereby achieving prolonged (over 3 weeks) activation of the IL-2 pathway.

Figure 5. NKTR-214

Initially, NKTR-214 showed promising results in early clinical trials, with an objective response rate (ORR) of 52.6% in first-line treatment of melanoma. However, the situation took a downturn after entering Phase III development. On March 14, 2022, Nektar/BMS jointly announced the failure of the Phase III PIVOT-12 study of NKTR-214 in combination with Opdivo for the treatment of unresectable or metastatic melanoma. NKTR-214 + Opdivo did not demonstrate superiority over Opdivo monotherapy in terms of progression-free survival (PFS), overall survival (OS), ORR, and other clinical endpoints.

On April 14, 2022, Nektar/BMS announced the termination of two clinical studies investigating NKTR-214 plus Opdivo for the treatment of renal cell carcinoma (RCC) and bladder cancer, both of which ended in failure. Consequently, they decided to terminate the global clinical development program for NKTR-214 plus Opdivo. Subsequently, Nektar gradually disclosed the results of four other clinical trials, all of which ended in failure.

IL-2 Fusion Protein

Denileukin Diftitox (Lymphir)

In March 2024, Citius Pharmaceuticals announced that the biologics license application (BLA) for its improved version of Denileukin Diftitox (Lymphir) has been accepted by the FDA. This application is intended for patients with relapsed or refractory cutaneous T-cell lymphoma (CTCL) who have undergone at least one systemic treatment. The PDUFA target date for this application is August 13, 2024.

Lymphir is a recombinant fusion protein that combines a fragment of diphtheria toxin with the IL-2R binding domain. By binding to the IL-2 receptors on the cell surface, Lymphir precisely delivers the diphtheria toxin, which inhibits protein synthesis in malignant T cells that enter the cell.

Figure 6. Lymphir 

This BLA application is primarily based on a pivotal Phase III study. The results of this trial demonstrated an ORR of 36.2%, as evaluated by an independent review committee (IRC). Efficacy analysis conducted by researchers revealed an ORR of 42.3%.

It is worth mentioning that Lymphir is a purified recombinant formulation of the anti-tumor drug Ontak. By refining the original formulation of Ontak, Lymphir possesses higher purity and biological activity. Ontak was developed by Eisai and was approved by the FDA in 1999. However, it was voluntarily withdrawn from the market later due to safety concerns such as vascular leakage.

Conslusion

The revival of IL-2-related drug development is a typical case of how basic research drives the advancement of the pharmaceutical industry. It is believed that in the future, with the progress of both basic and clinical research, the applications of IL-2 molecules in cancer, autoimmune diseases, and even other new disease areas will become increasingly widespread, providing humanity with powerful weapons to combat immune-related diseases.

References:
[1] Spolski, Rosanne et al. “Biology and regulation of IL-2: from molecular mechanisms to human therapy.” Nature reviews. Immunology vol. 18,10 (2018): 648-659. doi:10.1038/s41577-018-0046-y
[2] https://ir.nektar.com/news-releases/news-release-details/nektar-therapeutics-announces-phase-2-topline-data
[3] https://www.science.org/content/blog-post/nektar-reads-out-unfortunately
[4] https://www.cancernetwork.com/view/fda-accepts-resubmitted-bla-for-denileukin-diftitox-in-r-r-ctcl