As a T-cell growth factor, Interleukin 2 (IL-2) promotes immune action by acting on effector and memory T cells and also modulates immune tolerance via regulatory T cells (Treg cells). IL-2 was first discovered in the supernatant of activated T-cell cultures and was used in late development as the first approved immunotherapy to enhance T-cell immunity and NK cell populations in patients. Despite its efficacy, there are still obstacles to overcome with high doses of IL-2 therapy, with its short half-life (<15 min), high dose administration, and severe non-specific toxicity. Therefore, improving the design and mode of action of IL-2-based biologics has become a hot research topic.
1. IL-2 Signaling
IL-2 is a multifunctional cytokine secreted mainly by CD4-positive T cells. It regulates immune responses by binding to the IL-2 receptor (IL-2R). IL-2R is divided into two types, with high-affinity IL-2R consisting of three subunits, the α-chain (CD25), the β-chain (CD122) and the γ-chain (CD132). High-affinity IL-2R is mainly expressed on the surface of Treg cells, newly activated CD4-positive and CD8-positive effector T cells, some natural killer (NK) cells and NKT cells. The intermediate affinity IL-2R consists of two subunits (CD122 and CD132) and is mainly expressed on the surface of CD8-positive memory T cells and most NK cells. IL-2R signaling not only affects the proliferation of effector T cells, Treg cells, NK cells and other cells, but also shape their functional activity.
Lymphoid cell expression of high affinity IL-2R (right) and intermediate affinity IL-2R (left). Image source: Reference 
Although unmodified IL-2 was once approved for the treatment of renal cell carcinoma and melanoma, the use of high doses of IL-2 for cancer treatment faces multiple obstacles. The short half-life of natural IL-2 (<15 min) leads to very high doses of IL-2 to be infused intravenously, which can cause severe non-specific toxicity. Moreover, since IL-2 can simultaneously activate Treg in the tumor microenvironment, which normally acts as an immunosuppressive agent, it may counteract the anti-cancer immune response stimulated by IL-2. Therefore, current research is directed toward the development of engineered IL-2 analogues that activate only specific lymphocytes.
2. Engineered IL-2 Proteins
Earlier designs for IL-2 proteins focused on extending the half-life of IL-2 by fusion expression with other proteins (e.g., immunoglobulins or albumin) or fusion with specific antibodies that allow IL-2 to be targeted to specific tumor sites. Development of mutants of IL-2 with selective high affinity for IL-2R usually requires a reduction in the interaction of IL-2 with CD122 or CD25. There are four main classes of engineered IL-2 proteins: IL-2 muteins, PEGylated IL-2, IL-2-anti-IL-2 immune complexes and IL-2-CD25 fusion proteins.
2.1 IL-2 Muteins
IL-2 muteins alter specific amino acids on the surface of IL-2 protein by introducing genetic mutations. Crystal structure studies of the IL-2 and IL-2R complexes have determined the amino acids that play a key role in the binding of IL-2 to different IL-2R subunits. By modifying these amino acids, IL-2 mutants that tend to bind to high- or intermediate-affinity receptors can be generated.
For example, Amgen's efavaleukin alfa, is an investigational therapy that fuses IL-2, which carries a specific mutation, to the Fc end of an antibody, creating a fusion protein that not only has a longer half-life but also tends to bind to high-affinity receptors. As a result, it is able to promote the proliferation of Treg cells, thereby restoring immune homeostasis in patients with autoimmune diseases. Currently, it is in phase 2b clinical trials for the treatment of patients with systemic lupus erythematosus (SLE) and ulcerative colitis. Preliminary results obtained in SLE patients show that efavaleukin alfa selectively expands Treg cells in the circulation.
Image source: Amgen Official Website
2.2 PEGylated IL-2
Coupling polyethylene glycol (PEG) to the surface of IL-2 proteins can prolong their half-life. Moreover, by managing the location and amount of PEG coupling on the IL-2 surface, IL-2 analogues that tend to bind to high- or intermediate affinity receptors can be constructed. For example, bempegaldesleukin (NKTR-214), developed by Nektar Therapeutics, couples an average of six PEG chains near the interface where IL-2 binds to CD25. This PEG modification effectively blocks its binding to the high-affinity IL-2R receptor, allowing bempegaldesleukin to be more inclined to activate CD8-positive memory T cells and NK cells.
Bristol-Myers Squibb and Nektar have entered into a development agreement to evaluate the effect of bempegaldesleukin in combination with the anti-PD-1 antibody Opdivo in multiple tumor types. Five clinical trials with registrational potential are currently underway, although Bristol-Myers Squibb recently announced that the Phase 3 clinical trial of this combination therapy for melanoma did not meet the primary endpoint.
One of the drawbacks of some PEylated IL-2 (e.g., bempegaldesleukin) is that the PEGylated sites are not consistent across protein molecules, which may lead to a failure of some IL-2 proteins to selectively activate CD8-positive T cells and NK cells, thus compromising the efficacy of candidate therapies. To address this issue, some biotechnology companies have introduced PEG chain-coupled amino acids on the surface of IL-2. This approach is able to precisely locate the PEG coupling on the IL-2 surface, potentially generating more homogenous IL-2 analogues. Sanofi's SAR444245 (once known as THOR707) is a precisely PEGylated version of IL-2, where the PEG chain is attached to a novel amino acid inserted at a location on IL-2 that prevents it from engaging the alpha-receptor and binding to immune receptors that cause drug toxicities (IL-2R-alpha, CD25). Sanofi stated that this IL-2 analogue may have the potential to be the "best-in-class". It is currently undergoing multiple Phase 2 clinical trials in combination with PD-1 inhibitors or other anti-cancer therapies for a variety of different cancer types.
SAR444245 Introduction, image source: Sinofi official website
Biopharma PEG provides PEG derivatives for PEGylated IL-2 and a variety of supporting technical services. We will support you in all aspects of PEGylation from the laboratory R&D level to the scale-up level.
2.3 IL-2 immune complexes
The binding of IL-2 to anti-IL-2 monoclonal antibodies to form a complex is also a way to extend the half-life of IL-2 protein and to modulate the receptor binding selectivity of IL-2. By binding to specific epitopes of IL-2, monoclonal antibodies targeting IL-2 can block the binding of IL-2 to CD25 or CD122, thus conferring them the ability to bind selectively to high- or intermediate-affinity IL-2R.
For example, ANV419, a selective IL-2R agonist developed by Anaveon, fuses a monoclonal antibody targeting the IL-2 and CD25 binding sites to IL-2, thereby blocking the binding of IL-2 to CD25. This fusion protein still has the ability to bind to the intermediate affinity IL-2R receptor and therefore selectively activates CD8-positive memory T cells and NK cells. The binding to the antibody also prolongs its half-life.
Mechanism of Action of ANV419, image source: Anaveon official website
2.4 IL-2-CD25 Fusion Proteins
Another strategy to allow IL-2 to bind selectively to the intermediate affinity IL-2R receptor is to fuse IL-2 to soluble CD25, forming a fusion protein that can no longer bind to CD25 on the cell surface, thus blocking binding to the high affinity IL-2R.
Alkermes has developed nemvaleukin alfa (ALKS 4230), which represents such a fusion protein. It activates CD8-positive memory T cells and NK cells without activating Treg cells by selectively binding to the medium-affinity IL-2R receptor. This investigational therapy has been granted two Fast Track status by the FDA for use in combination with anti-PD-1 antibodies for the treatment of platinum-containing chemotherapy-resistant ovarian cancer, and mucosal melanoma, respectively. It is in Phase 2 and Phase 3 clinical development.
Mechanism of Action of nemvaleukin alfa, image source: Reference 
3. Pros And Cons Of Different Engerneered IL-2 Proteins
These engineered IL-2 protein modifications also have their own pros and cons. For example, one potential concern with IL-2 muteins is that the introduced genetic mutation may cause the resulting IL-2 muteins to be perceived as a foreign protein by the body's immune system, triggering an immune response against the IL-2 mutant and thus reducing the efficacy of potential therapies.
PEGylated IL-2, IL-2 immune complex and IL-2-CD25 fusion protein could theoretically confer selectivity to IL-2 without introducing mutations, thereby reducing the immunogenicity of candidate therapies. However, they also have their own shortcomings. An uncertain site of PEG modification may lead to heterogeneity of the product and compromise the overall efficacy. The IL-2 immune complex may release free IL-2 in the circulation, leading to stimulation of other immune cells that do not intend to be stimulated.
While there are multiple strategies to engineer IL-2, giving them the ability to selectively activate anti-cancer immune responses or inhibit overactive immune responses, which strategy is safer and more effective still needs to be validated in subsequent clinical trials. With several IL-2 analogues currently in clinical development, we will see in the coming years whether these advances in IL-2 modification will lead to effective anti-cancer and innovative therapies for the treatment of autoimmune diseases.
 Hernandez et al.,(2022). Engineering IL-2 for immunotherapy of autoimmunity and cancer. Nature Reviews Immunology,https://doi.org/10.1038/s41577-022-00680-w
 Amgen Business Review. Retrieved March 9, 2022, from https://investors.amgen.com/static-files/6d823d7d-2fd1-405a-8c0e-22aa91bee682
 Nektar Therapeutics Corporate Presentation. Retrieved March 9, 2022, from https://ir.nektar.com/static-files/7818281a-1882-4952-9ebc-7680643f109a
 Sanofi Oncology ASCO Event. Retrieved March 9, 2022, from https://www.sanofi.com/-/media/Project/One-Sanofi-Web/Websites/Global/Sanofi-COM/Home/common/docs/investors/2021_06_04_ASCO_Slides_FINAL.pdf
 Anaveon. Retrieved March 9, 2022, from https://anaveon.com/our-approach/
 Alkermes to Present Data on Nemvaleukin Alfa at the Society of Gynecologic Oncology 2022 Annual Meeting on Women's Cancer. Retrieved March 9, 2022, from https://www.prnewswire.com/news-releases/alkermes-to-present-data-on-nemvaleukin-alfa-at-the-society-of-gynecologic-oncology-2022-annual-meeting-on-womens-cancer-301492162.html
 Lopes, et al., (2021). Pharmacokinetics and Pharmacodynamic Effects of Nemvaleukin Alfa, a Selective Agonist of the Intermediate-Affinity IL-2 Receptor, in Cynomolgus Monkeys. Journal of Pharmacology and Experimental Therapeutics, https://doi.org/10.1124/jpet.121.000612
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