The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-β1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-β bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a “QKS” site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors.

To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-β1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone.

In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

新型抗 PD-L1/TGFBR2-ECD 融合蛋白 (BR102) 包含抗 PD-L1 抗体 (HS636)，该抗体在重链 C 端与 TGF-β1 受体 Ⅱ 胞外域 (TGFBR2-ECD) 融合)，并且可以隔离免疫抑制肿瘤微环境中的 PD-1/PD-L1 通路和 TGF-β 生物活性。在 TGFBR2-ECD 野生型融合蛋白 (BR102-WT) 的表达中，证实了在位于胞外域 N 末端的“QKS”位点的蛋白水解切割诱导了一个 50 kDa 剪辑的物种，这导致形成IgG样剪切。基质金属蛋白酶 9 被确定与 BR102-WT 消化有关。此外，

为了避免蛋白水解降解，进行了消除蛋白酶敏感的氨基酸基序并引入潜在的糖基化。三个敏感的基序发生了突变，并且剪辑的水平受到了强烈的限制。突变候选物对 hPD-L1 和 hTGF-β1 以及高度纯化的 BR102-WT2 表现出相似的结合亲和力。此外，突变体在裂解物孵育反应和血浆稳定性测试中表现出比BR102-WT2更显着的蛋白水解抗性。此外，与单独使用抗 PD-L1 抗体相比，双功能候选 Mu3 在 MC38/hPD-L1 轴承模型中显示出附加的抗肿瘤作用。

总之，在本研究中，BR102-WT 的蛋白酶敏感性特征得到了很好的表征，并进行了有效的优化。候选BR102-突变体在药物稳定性方面表现出先进的成药性，并表现出理想的抗肿瘤活性。