A novel alphaPD-1/alphaVEGF/IL-21v tri-specific fusion protein exhibits potent antitumor efficacy beyond alphaPD-1/alphaVEGF bispecific therapy

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 alphaPD-1 with alphaVEGF monoclonal antibodies (mAbs) or alphaPD-1/alphaVEGF bispecific antibodies (bsAbs) have demonstrated improved efficacy relative to alphaPD-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). alphaPD-1/alphaVEGF/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 alphaPD-1, enhance tumor-infiltrating lymphocytes (TILs) via alphaVEGF, and promote effector/memory T cells via IL-21v. The alphaPD-1/alphaVEGF/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-1 high T cells treated with alphaPD-1/alphaVEGF/IL-21v secreted markedly higher IFN-gamma and granzyme B than with alphaPD-1/alphaVEGF. Moreover, alphaPD-1/alphaVEGF/IL-21v promoted proliferation of CD8 + IFN-gamma + Granzyme B + effector T cells and improved CD8⁺T/Treg ratio under in vitro TME-mimicking conditions, whereas alphaPD-1/alphaVEGF did not produce these effects. Importantly, under resting, peripheral-mimicking conditions, alphaPD-1/alphaVEGF/IL-21v induced minimal immune cell proliferation and low cytokine release. In ICI-resistant MC38 tumor model, alphaPD-1/alphaVEGF/IL-21v demonstrated superior antitumor efficacy compared with alphaPD-1 or alphaPD-1/alphaVEGF 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, alphaPD-1/alphaVEGF/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 alphaPD-1 or alphaPD-1/alphaVEGF therapy.