Mohamedali Khalid A, Aguirre Brian, Lu Cheng-Hsiang, Chandla Anubhav, Kejriwal Nidhi, Liu Lucia, Chan Ann M, Cheung Lawrence H, Kok SuYin, Duarte Sergio, Alvarez de Cienfuegos Ana, Casero David, Rosenblum Michael G, Wadehra Madhuri
Experimental Therapeutics, University of Texas MD Anderson Cancer Center Division of Cancer Medicine, Houston, Texas, USA.
Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USA.
J Immunother Cancer. 2024 Dec 22;12(12):e008891. doi: 10.1136/jitc-2024-008891.
Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an innovative therapeutic approach to specifically deliver a deadly payload to target cells. Epithelial membrane protein-2 (EMP2) is highly expressed in invasive breast cancer (BC), including triple-negative BC (TNBC), and represents an attractive therapeutic target.
We designed a novel fusion protein (GrB-Fc-KS49) composed of an active GrB fused to an anti-EMP2 single-chain antibody tethered through the immunoglobulin G heavy chain (Fc) domain. We assessed the construct's GrB enzymatic activity, anti-EMP2 binding affinity, and cytotoxicity against a panel of BC cells. The construct's pharmacokinetics (PK), toxicity profile, and in vivo efficacy were also evaluated.
GrB-Fc-KS49 exhibited comparable GrB enzymatic activity to commercial GrB, as well as high affinity to an EMP2 peptide, with the dissociation constant in the picomolar range. The fusion protein rapidly internalized into EMP2+cancer cells and showed in vitro cytotoxicity to cell lines expressing surface EMP2, with half-maximal cytotoxicity (IC) values below 100 nM for most positive lines. Ex vivo stability at 37°C indicated a half-life exceeding 96 hours while in vivo PK indicated a biexponential plasma clearance, with a moderate initial clearance (tα=18.4 hours) and a much slower terminal clearance rate (tβ=73.1 hours). No toxicity was measured in a Chem16 panel between the control and the GrB-Fc-KS49. In vivo, the GrB-Fc-KS49 showed efficacy against a TNBC syngeneic (4T1/) mouse model, reducing tumor volume and cell proliferation and increasing cell death compared with controls. Treatment using an EMT6 mouse model confirmed these results. In addition to a significant impact on cell proliferation, GrB-Fc-KS49 treatment also resulted in a dramatic increase of tumor-infiltrating CD45+ cells and redistribution of tumor-associated macrophages. Transcriptomic analysis of tumors post-treatment confirmed the remodeling of the immune tumor microenvironment by the GrB-Fc-KS49 immunotoxin.
GrB-Fc-KS49 showed high specificity and cytotoxicity towards EMP2-positive cells. In vivo, it reduced tumor burden and increased the recruitment of immune cells into the tumor, suggesting that GrB-Fc-KS49 is a promising therapeutic candidate against BC.
颗粒酶B（GrB）是一种关键效应分子，在免疫监视过程中由细胞毒性T淋巴细胞和自然杀伤细胞释放，以诱导细胞死亡。融合蛋白和免疫缀合物是一种创新的治疗方法，可将致命的药物特异性递送至靶细胞。上皮膜蛋白2（EMP2）在包括三阴性乳腺癌（TNBC）在内的浸润性乳腺癌（BC）中高表达，是一个有吸引力的治疗靶点。
我们设计了一种新型融合蛋白（GrB-Fc-KS49），它由活性GrB与通过免疫球蛋白G重链（Fc）结构域连接的抗EMP2单链抗体融合而成。我们评估了该构建体的GrB酶活性、抗EMP2结合亲和力以及对一组BC细胞的细胞毒性。还评估了该构建体的药代动力学（PK）、毒性特征和体内疗效。
GrB-Fc-KS49表现出与市售GrB相当的GrB酶活性，以及对EMP2肽的高亲和力，解离常数在皮摩尔范围内。该融合蛋白迅速内化到EMP2+癌细胞中，并对表达表面EMP2的细胞系显示出体外细胞毒性，大多数阳性细胞系的半数最大细胞毒性（IC）值低于100 nM。在37°C下的体外稳定性表明半衰期超过96小时，而体内PK表明血浆清除呈双指数形式，初始清除率适中（tα=18.4小时），终末清除率慢得多（tβ=73.1小时）。在Chem16检测中，对照组和GrB-Fc-KS49之间未检测到毒性。在体内，GrB-Fc-KS49对TNBC同基因（4T1/）小鼠模型显示出疗效，与对照组相比，可减少肿瘤体积和细胞增殖，并增加细胞死亡。使用EMT6小鼠模型进行的治疗证实了这些结果。除了对细胞增殖有显著影响外，GrB-Fc-KS49治疗还导致肿瘤浸润性CD45+细胞显著增加以及肿瘤相关巨噬细胞的重新分布。治疗后肿瘤的转录组分析证实了GrB-Fc-KS49免疫毒素对免疫肿瘤微环境的重塑作用。
GrB-Fc-KS49对EMP2阳性细胞表现出高特异性和细胞毒性。在体内，它减轻了肿瘤负担，并增加了免疫细胞向肿瘤的募集，表明GrB-Fc-KS49是一种有前景的抗BC治疗候选药物。
