Abstract 4341: A novel αPD-1/αVEGF/IL-21v tri-specific fusion protein exhibits potent antitumor efficacy beyond αPD-1/αVEGF bispecific therapy

Abstract Immune checkpoint inhibitors (ICIs) have been widely used for multiple malignancies. Nevertheless, response rates to ICIs remain limited across several tumor types, largely due to insufficient T cell infiltration and the inability to sustain long-term antitumor immunity within the tumor microenvironment (TME). Combinations of αPD-1 with αVEGF monoclonal antibodies (mAbs) or αPD-1/αVEGF bispecific antibodies (bsAbs) have demonstrated improved efficacy relative to αPD-1 monotherapy across multiple cancers. Despite such advances, strategies that not only enhance effector T cell accumulation within the TME but also promote sustained antitumor immune responses are essential to achieve therapeutic benefit in ICI-resistant or -refractory settings. Interleukin-21 (IL-21) enhances CD8+ T cell activation and memory T cell differentiation while limiting Treg expansion, supporting durable antitumor immunity. However, its broad off-target activity and short half-life limit clinical utility, motivating the development of TME-selective IL-21 variants (IL-21v). αPD-1/αVEGF/IL-21v is a tri-specific fusion protein comprising anti-PD-1, anti-VEGF, and an IL-21v engineered to minimize peripheral toxicity while sustaining T cell activation within the TME. This construct was designed to sustain T cell activation via αPD-1, enhance tumor-infiltrating lymphocytes (TILs) via αVEGF, and promote effector/memory T cells via IL-21v. The αPD-1/αVEGF/IL-21v exhibited PD-1/PD-L1 blocking activity comparable to pembrolizumab and VEGF/VEGFR blockade similar to bevacizumab. In PD-1+/PD-1- HuT78 cells, it induced PD-1-dependent STAT3 activation with ∼5,000-fold selectivity for PD-1+ cells. Consistently, PD-1high T cells treated with αPD-1/αVEGF/IL-21v secreted markedly higher IFN-γ and granzyme B than with αPD-1/αVEGF. Moreover, αPD-1/αVEGF/IL-21v promoted proliferation of CD8+IFN-γ+Granzyme B+ effector T cells and improved CD8+T/Treg ratio under in vitro TME-mimicking conditions, whereas αPD-1/αVEGF did not produce these effects. Importantly, under resting, peripheral-mimicking conditions, αPD-1/αVEGF/IL-21v induced minimal immune cell proliferation and low cytokine release. In ICI-resistant MC38 tumor model, αPD-1/αVEGF/IL-21v demonstrated superior antitumor efficacy compared with αPD-1 or αPD-1/αVEGF treatment, without adverse effects. Animals achieving complete responses did not relapse upon tumor re-challenge. This long-lasting efficacy was associated with an increased intratumoral CD8+T/Treg ratio and expansion of memory T cell populations within tumors rather than in peripheral blood. In conclusion, αPD-1/αVEGF/IL-21v is a novel TME-selective tri-specific fusion protein that enhances TIL recruitment and amplifies effector/memory T cell populations, offering therapeutic potential beyond αPD-1 or αPD-1/αVEGF therapy. Citation Format: Suna Kim, Sunjung Cho, Hyojoo Bang, So Woon Kim, Yongjun Jung, Seokchan Kang, Suyoon Kim, Mi-Seong Kim, Jun-Eui Park, Young Jin Park, Sungyoub Jung. A novel αPD-1/αVEGF/IL-21v tri-specific fusion protein exhibits potent antitumor efficacy beyond αPD-1/αVEGF bispecific therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4341.