Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep).
Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep).
复制标题
作者：
Ngambenjawong C;Gustafson HH;Pineda JM;Kacherovsky NA;Cieslewicz M;Pun SH
肿瘤相关巨噬细胞（tam）是几种癌症肿瘤微环境的主要基质成分。由于tam具有促进癌细胞增殖、血管生成和转移的作用，因此它是辅助癌症治疗的潜在靶点。我们之前发现了一种M2巨噬细胞靶向肽（M2pep），该肽成功地用于在CT-26结肠癌模型中靶向并向M2样tam传递促凋亡的KLA肽。然而，使用M2pep在体内靶向tam的有效性受到其血清稳定性差和低结合亲和力的限制。在这项研究中，我们合成了M2pep衍生物，目的是提高血清稳定性和结合亲和力。M2pepBiotin的血清稳定性评估证实其快速降解归因于n端外溶性裂解和W10/W11和S16/K17位点的内溶性裂解。M2pepBiotin的n端乙酰化保护肽段免受外溶降解，而W10w和K(17,18,19) K取代则能够在各自的裂解位点有效地保护肽段的内溶降解。然而，W10位点的氨基酸变化没有导致该位点的蛋白酶抗性和结合活性的保留。因此，对M2pep的环化进行了研究。环化的M2pep更好地抵抗血清降解，而不影响与M2巨噬细胞的结合活性。在血清稳定性优化过程中，我们还发现K9R和W10Y的取代显著增强了M2pep的结合亲和力。在小鼠血清中预先培养的不同M2pep类似物的体外结合研究中，经过K9R和W10Y修饰的环M2pep（环M2pep(RY)）由于其改善的血清稳定性，随着时间的推移，对M2巨噬细胞的结合活性保持最高。最后，我们评估了sulo - cy5标记的M2pep和环M2pep（RY）在CT-26和4T1乳腺癌模型中的体内积累。在m2样tam中，环M2pep（RY）在肿瘤定位和选择性积累方面都优于M2pep。总之，我们报告环M2pep（RY）作为我们的M2pep类似物，具有改善的血清稳定性和M2巨噬细胞结合活性。在两种肿瘤模型中证明了其作为体内M2样tam靶向剂的增强效用，预计将适用于其他肿瘤模型或M2巨噬细胞相关疾病模型。
Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep(RY) outperformed M2pep in both tumor localization and selective accumulation in M2-like TAMs. In conclusion, we report cyclic M2pep(RY) as our lead M2pep analog with improved serum stability and M2 macrophage-binding activity. Its enhanced utility as an in vivo M2-like-TAM-targeting agent was demonstrated in two tumor models, and is expected to be applicable for other tumor models or in models of M2 macrophage-related diseases.