IRCM, INSERM U1194, University of Montpellier, ICM, Montpellier, France.
Université de Tours - INRAE, UMR1282, Infectiologie et Santé Publique (ISP), équipe BioMédicaments Anti-Parasitaires (BioMAP), Tours, France.
J Immunother Cancer. 2024 Jan 30;12(1):e007135. doi: 10.1136/jitc-2023-007135.
Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC.
Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc) or prevent (F1M1-Fc) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide.
Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc displayed higher antitumor activity than F1M1, whereas F1M1-Fc was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc antitumor activity, demonstrating their key role. F1M1-Fc inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc improved paclitaxel and enzalutamide therapeutic efficacy without toxicity.
F1M1-Fc is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.
三阴性乳腺癌（TNBC）的预后较差。为了增强针对 TNBC 的抗体诱导的自然杀伤（NK）细胞抗肿瘤活性的免疫疗法正在出现，因为 TNBC 通常具有免疫原性。天冬氨酸蛋白酶组织蛋白酶 D（cath-D）是一种与肿瘤细胞相关的细胞外蛋白，具有促进肿瘤的活性，并且是 TNBC 预后不良的标志物，它是一种用于诱导 NK 细胞介导的抗体依赖性细胞毒性（ADCC）的基于抗体的治疗的主要靶标。本研究调查了 Fc 工程化的抗 cath-D 抗体是否触发 ADCC、它们对肿瘤疗效和肿瘤浸润性 NK 细胞的影响，以及它们与 TNBC 联合治疗的相关性。
通过 Western blot、免疫荧光和免疫组织化学评估 TNBC 样本中的 cath-D 表达和定位。通过 ELISA 分析人源抗 cath-D F1M1 和 Fc 工程化抗体变体（增强（F1M1-Fc）或阻止（F1M1-Fc）与 CD16a 结合）与人源和鼠源 cath-D 的结合，通过表面等离子体共振和流式细胞术分析与 CD16a 的结合。通过流式细胞术研究 NK 细胞的激活，通过乳酸脱氢酶释放研究 ADCC。使用裸鼠中的 TNBC 细胞异种移植研究 F1M1 Fc 变体的抗肿瘤功效。通过免疫表型分析和 RT-qPCR 分析 MDA-MB-231 细胞异种移植中的 NK 细胞募集、激活和细胞毒性活性。使用抗神经节苷脂 GM1 抗体耗尽 NK 细胞。在 TNBC 患者来源异种移植（PDX）和 TNBC SUM159 细胞异种移植中评估 F1M1-Fc 的抗肿瘤作用，并与紫杉醇或恩扎鲁胺联合评估。
TNBC 细胞表面上的 cath-D 表达可用于诱导 ADCC。F1M1 Fc 变体识别人源和鼠源 cath-D。F1M1-Fc 在体外激活 NK 细胞，并比 F1M1 更有效地诱导针对 TNBC 细胞和癌相关成纤维细胞的 ADCC。F1M1-Fc 无效。在 MDA-MB-231 细胞异种移植模型中，F1M1-Fc 比 F1M1 具有更高的抗肿瘤活性，而 F1M1-Fc 的效果较差，反映了体内 Fc 依赖性机制的重要性。F1M1-Fc 在 MDA-MB-231 细胞异种移植中触发肿瘤浸润性 NK 细胞募集、激活和细胞毒性活性。NK 细胞耗竭会损害 F1M1-Fc 的抗肿瘤活性，证明了它们的关键作用。F1M1-Fc 抑制 SUM159 细胞异种移植和两个 TNBC PDX 的生长。在联合治疗中，F1M1-Fc 改善了紫杉醇和恩扎鲁胺的治疗效果，而没有毒性。
F1M1-Fc 是一种有前途的 TNBC 免疫疗法，可与包括化疗或抗雄激素在内的常规方案联合使用。
