Background
Interleukin-2 (IL-2) is an agonist that plays a crucial role in cancer immunotherapy. High-dose recombinant IL-2 (Proleukin®/aldesleukin) is the first approved cytokine-based immunotherapy for metastatic melanoma (MM) and renal cell carcinoma (RCC). IL-2's short half-life, cumbersome high-dose dosing regimens, and severe dose-limiting toxicities, including capillary leak syndrome and cardiopulmonary, hepatic, and renal toxicities, have hindered its widespread use in cancer immunotherapy. Furthermore, high-dose IL-2 not only expands anti-tumor effector cells but also serves as a potent growth factor for immunosuppressive regulatory T cells (Tregs), which are often associated with poor prognosis in cancer patients.
On July 23, 2024, researchers published a study in MAbs titled "Discovery and development of ANV419, an IL-2/anti-IL-2 antibody fusion protein with potent CD8+ T and natural killer cell-stimulating capacity for cancer immunotherapy". In this study, researchers designed an IL-2 agonist, ANV419, which is an IL-2/anti-IL2 antibody fusion protein designed to selectively activate IL-2 receptor βγ (IL-2 Rβγ) by sterically hindering IL-2 binding to IL-2Rα. The agonist is selective for IL-2 Rβγ, has an extended half-life, moderate toxicity, and can effectively inhibit tumor growth in syngeneic mouse models.
IL-2 Antibody/IL-2 Fusion Protein Development
A humanized anti-IL-2 antibody, NARA1, was developed. The N- and C-termini of IL-2 were attached before and after helix αA and αD, respectively, to create a circularly arranged IL-2 (IL-2CP). This circularly arranged IL-2 molecule was fused to the light chain variable region (LCDR1) of a high-affinity humanized anti-IL-2 antibody to form an antibody-cytokine fusion protein. The novel N- and C-termini of IL-2CP were linked to residues Y27d and D30 of the antibody's LCDR1, respectively, via glycine-serine linkers.
Candidate fusion proteins were generated using three antibodies, A, B, and C, and a set of specific glycine-serine linkers. Antibody A was found to bind to IL-2 with high affinity. The high-affinity IL-2-binding Antibody A fusion protein sterically blocked the binding of cyclic IL-2 to IL-2 Rα, even with long linkers. The study found that high-affinity antibody fusions maintained selectivity over a wide range of linker lengths, while non-affinity antibody fusions were non-selective even with very short linkers. Fusion proteins of IL-2 and Antibody A were constructed with GGG and GGGG linkers, and the fusion protein was named ANV419.
By solving the structure of ANV419, it was found that the binding site of antibody A and IL-2 overlaps with most of the IL-2 residues on helix αB that overlaps with IL-2 Rα.
Selectivity of ANV419 for IL-2 Rβγ-expressing cells
In vitro stimulated mouse splenocytes, ANV419 reduced the efficacy of pSTAT5 phosphorylation in Tregs, but its activation of CD8+ T cells and NK cells was comparable to that of conventional IL-2. In vivo, a single intravenous administration of ANV419 induced the proliferation of CD8+ T cells and NK cells in a dose-dependent manner, while having little effect on Treg cells. In contrast, the antibody C fusion protein exhibited a strong proliferative effect on Treg cells.
ANV419 can inhibit tumor growth
H22 hepatocellular carcinoma tumors responded strongly to ANV419 monotherapy, and additional tumor growth inhibition (TGI) was observed when ANV419 was combined with PD-1 or CTLA-4 CPIs. ANV419 monotherapy also achieved significant TGI in the partially CPI-responsive Renca renal cell carcinoma model, with only limited growth retardation observed in the less immunogenic B16F10 melanoma model. ANV419 treatment in Renca significantly enhanced tumor infiltration by CD8+ T cells and NK cells, and Treg numbers and proliferation were not increased by ANV419.
Administration of ANV419 also resulted in an increase in NKT cells (a cell type expressing IL-2Rβγ), but this was not observed in CD4+ T cells, which express low levels of IL-2R at steady state and are less responsive to IL-2, as well as in B cells and macrophages, which completely lack IL-2R expression.
ANV419 and its IL-2R signaling potency and kinetics
After stimulation with ANV419 and IL-2, CD8+ T cells and NK cells showed similar EC50 values, but in Tregs, a decrease in IL-2R-mediated signaling potency was observed after treatment with ANV419 compared to IL-2, indicating that ANV419 is more selective for CD8+ T cells and NK cells than for Tregs in human cells. CD8+ T cells and NK cells stimulated with ANV419 exhibited pSTAT5 kinetics comparable to IL-2, while STAT5 phosphorylation by IL-15 tended to be slightly faster than that by ANV419 or IL-2. Therefore, in CD8+ T cells and NK cells, the IL-2R signaling potency and kinetics of ANV419 are comparable to those of IL-2.
ANV419 enhances NK cell killing ability and ADCC
The study found that treating NK cells with ANV419 did not alter the expression of NK cell receptors on their surface, but resulted in a higher activation state of the NK cells. ANV419-treated NK cells showed increased cytotoxicity against NCI-H929 myeloma and HCC1954 epithelial breast cancer target cells. Co-incubation of NK cells with ANV419 and either daratumumab or trastuzumab enhanced the activity of the antibodies. Combining it with trastuzumab, an antibody with ADCC activity, showed an additive effect in inhibiting tumor growth. These results suggest that ANV419 enhances NK cell cytotoxicity and ADCC.
ANV419 half-life and pharmacological analysis
Through pharmacology and toxicology studies of ANV419 in crab-eating macaques, it was found that ANV419 has a prolonged half-life and promotes the proliferation of CD8+ T cells and NK cells in crab-eating macaques.
ANV419 induces a transient dose-dependent inflammatory response, but does not trigger the significant adverse reactions of high-dose IL-2 due to its feedback mechanism at high doses.
Summary and Outlook
This study overcomes the shortcomings of recombinant IL-2 through protein engineering. Based on the fusion of IL-2 with the high-affinity anti-IL-2 antibody A, a circular arrangement strategy is used to retain the antigen/antibody interaction while still allowing the binding and activation of IL-2 Rβγ to form the fusion protein ANV419. This agonist is selective for IL-2 Rβγ and can inhibit tumor growth and enhance the infiltration and expansion of CD8+ T cells and NK cells when used alone or in combination with CPIs. ANV419 has a prolonged half-life, moderate toxicity, and can effectively inhibit tumor growth in syngeneic mouse models. This study provides a theoretical basis for the clinical development of ANV419 in solid tumors and hematological malignancies, either as a monotherapy or in combination with checkpoint inhibitors or ADCC induction therapy.
ANV419 is currently in Phase 1/2 clinical development and may offer a wider therapeutic window for cancer patients than interleukins.
