Background

Antibody-cytokine fusion proteins (also known as immunocytokines) deliver these immunostimulatory payloads to tumor lesions, significantly broadening the therapeutic window of cytokine therapy. Furthermore, the combination of the antibody and cytokine components of immunocytokines will produce synergistic antitumor effects. However, immunocytokines may be captured by circulating cognate receptors before reaching target cells (the so-called "cytokine sink" effect). Therefore, developing practical strategies to overcome the safety challenges associated with immunocytokines and accelerate their clinical application is crucial.

On April 25, 2024, Lu Huili and other teams from Shanghai Jiao Tong University published a research result titled "Next-generation anti-PD-L1/IL-15 immunocytokine elicits superior antitumor immunity in cold tumors with minimal toxicity" in Cell Reports Medicine.

Researchers have developed a conditionally activated anti-PD-L1/ IL-15 (LH05), a prodrug that leverages steric hindrance to influence IL-15 activity. This immunocytokine is characterized by reduced toxicity and enhanced tumor targeting. It converts "cold" tumors into "hot" tumors, thereby eliciting remarkable anti-tumor immunity. The results provide compelling proof of concept for the development of next-generation immunocytokines and significantly contribute to current immunotherapy knowledge and strategies. 

Features of LH01

IL-15 is a highly attractive immunostimulatory cytokine, renowned for its remarkable activity in treating various cancer types. Immunocytokines with IL-15 as a payload have shown great promise in clinical applications, including KD033, which targets PD-L1, and BJ-001, which targets integrins. Previously, an anti-PD-L1/IL-15 immunocytokine (LH01) was developed that could overcome anti-PD-L1 resistance and elicit both innate and adaptive immune responses. However, LH01 also induced systemic toxicity similar to IL-15.

(Data source: Shi W, et al. Mol Ther. 2023)

Design of anti-PD-L1/IL-15 (LH05)

Previous studies have demonstrated that the anti-PD-L1/IL-15 immunocytokine (LH01) has potent antitumor efficacy in both syngeneic and xenograft models. However, dose-limiting toxicity has hindered the efficacy of LH01 in treating cold tumors. To improve efficacy and reduce toxicity, LH03 was designed by fusing IL-15 to the C-terminus of anti-PD-L1 and the N-terminus of a sushi domain via a flexible linker to reduce IL-15 activity. While LH03 improved safety, it lacked significant antitumor efficacy. Consequently, LH05 was designed by incorporating a protease-cleavable linker between the antibody and the ILR, which exploits the steric hindrance caused by the Fc fragment and sushi domain to mask IL-15 activity.

LH05 active switch

The cleavable linker acts as a switch for IL-15 activity. Prior to cleavage, IL-15 is shielded by the combined forces of the Fc fragment and the su-shi domain. ILR cleavage releases IL-15, restoring its anti-tumor activity. Compared to LH01, LH05 significantly reduced its in vitro proliferative activity, but after cleavage with urokinase-type plasminogen activator (uPA), its activity was restored by over 30-fold. Furthermore, LH05's in vitro binding affinity for human PD-L1 was similar to that of anti-PD-L1 antibodies and LH01, demonstrating that the anti-PD-L1 portion of LH05 remains intact, while the ILR portion is preferentially released within the TME to restore IL-15 activity.

LH05 safety in vivo

LH05 's significantly improved safety compared to LH01, mice were treated with PBS, LH01, and LH05. After two LH01 treatments, all mice experienced a dramatic weight loss and ultimately died within six days. However, mice treated with LH05 showed no mortality or weight loss, even after six injections. Compared to PBS treatment, LH05 treatment did not significantly increase the proportion of CD8+ T cells in the spleen or the number of CD8+ T cells in peripheral blood. Unlike LH01, LH05 did not significantly trigger the production of cytokines such as IFN-g and IL-6, further demonstrating that the risk of LH05-induced systemic toxicity is significantly reduced.

LH05 half-life is prolonged and tumor targeting distribution is improved

Due to the ubiquitous expression of their cognate receptors, immune cytokines may rapidly disappear from the circulation before reaching tumor tissue. LH05 has a reduced affinity for IL-15, but its kinetics are superior to LH01, and its half-life is prolonged. Studies have found that LH05 is primarily cleaved in tumor tissue, with less cleavage in normal tissue, resulting in higher concentrations of LH05 in tumor tissue.

LH05 induces innate and adaptive immune responses

Flow cytometry analysis revealed that LH05 significantly induced the generation of effector memory CD8+ T cells within the tumor, while central memory CD8+ T cells were more prevalent in the spleen. Furthermore, the proportion of naive CD8+ T cells in the spleen was higher than in the tumor. Compared with LH01, LH05 treatment resulted in an increase in CD8+ tumor-infiltrating lymphocytes (TILs). LH05 treatment also significantly elevated tumor-associated activated NK cells, potentially contributing to the enhanced anti-tumor efficacy of LH05. LH05 can activate both CD8+ T cells and NK cells to suppress tumors.

Anti-tumor effect of LH05

Using the RM-1 tumor-bearing mouse model (a typical "cold tumor" model), researchers found that NK cells and CD8+ cells are essential for anti-tumor activity. Using the drug FTY720, they found that inhibiting lymph node output completely abolished the efficacy of LH05. LH05's anti-tumor activity primarily relies on CD8+ T and NK cells, which infiltrate the TME from the circulation. Furthermore, LH05 may modulate the TME by altering the balance of chemokine and receptor signaling, thereby achieving an anti-tumor immune response. CXCL9 and CXCL10 are key factors that promote immune cell migration into the TME and add "heat" to the tumor. LH05 treatment resulted in a significant increase in Cxcl9 and Cxcl10 expression. IL-15 promotes immune cell function within the tumor through a cytokine network involving XCL1, IFN-g, CXCL9, and CXCL10. In summary, after reaching the TME, LH05 activates the immune response through ILR release, leading to the recruitment of CD8+ T and NK cells, promoting their expansion and cytotoxicity, and inducing the secretion of Th-1 type cytokines, thereby exerting a powerful anti-tumor immune effect.

Meanwhile, researchers used another "cold" tumor model, U251 glioblastoma, and found that treatment with LH05 inhibited tumor growth and reduced tumor proliferation and metastasis. Furthermore, researchers generated LH06 by replacing the urokinase substrate linker in LH05 with an MMP-2/14 substrate linker. Combination therapy with LH06 and the oncolytic poxvirus V200 demonstrated a strong synergistic antitumor effect in an advanced B16-F10 cold tumor model. Compared to IgG treatment, LH06 increased CD8+ T cells but not regulatory T cells (Tregs) in the tumor. LH06 and anti-PD-1 monotherapy significantly inhibited lung tumor metastasis at similar levels , with the combination therapy demonstrating a synergistic effect superior to either monotherapy. LH06 has considerable potential for treating advanced and metastatic tumors.

Summarize

This study developed an anti-PD-L1/IL-15 prodrug (LH05). LH05 innovatively masks IL-15 through steric hindrance, mitigating IL-15's "cytokine sink" effect and reducing the systemic toxicity associated with wild-type anti-PD-L1/IL-15. Furthermore, after specific proteolytic cleavage in the tumor microenvironment, LH05 releases an active IL-15 superagonist, exerting potent antitumor effects. The antitumor effects of LH05 rely on increased infiltration of CD8+ T cells and natural killer cells by stimulating the chemokines CXCL9 and CXCL10, thereby converting cold tumors into hot tumors.