目的
使用[ 68 Ga]Ga-PentixaFor-PET 对CXCR4 表达进行非侵入性成像的临床成功保证了将靶向概念扩展到传统闪烁扫描/SPECT 的成本较低和普遍可用性。为此，我们基于PentixaFor支架开发并比较评估了一系列99m Tc标记的环状五肽。
方法
具有不同 4-氨基苯甲酸 (Abz)-D-Ala-D-Arg-aa 3连接子 ( L1–L6 ) 的 6 个 mas 3缀合的 CPCR4 类似物以及L6- CPCR4 的相应 HYNIC- 和 N 4 - 类似物通过标准 SPPS 合成。使用Jurkat T细胞淋巴瘤细胞和[ 125 I]FC-131作为放射性配体进行竞争性结合研究（IC 50和IC 50 inv）。使用 hCXCR4 过表达 Chem-1 细胞研究内化动力学。使用带有 CB17/SCID 小鼠的 Jurkat 异种移植物进行生物分布研究和小动物 SPECT/CT 成像（注射后 1 小时）。根据临床前结果，[ 99m Tc]Tc-N 4 - L6- CPCR4 ([ 99m Tc]Tc-PentixaTec) 被选择用于早期转化为人类环境。五名患有血液恶性肿瘤的患者接受了[ 99m Tc]Tc-N 4 - L6 -CPCR4 SPECT/平面成像和个体剂量测定。
结果
在六种mas 3缀合肽中，mas 3 -L6 -CPCR4 (mas 3 -dap-ra-Abz-CPCR4) 显示出最高的CXCR4 亲和力(IC 50 = 5.0 ± 1.3 nM)。与 N 4 (N 4 - L6 -CPCR4) 缀合进一步将 hCXCR4 亲和力提高至 0.6 ± 0.1 nM。 [ 99m Tc]Tc-N 4 - L6- CPCR4 还显示出化合物最有效的内化（2 小时总细胞活性的 97%）和最高的肿瘤积累（8.6 ± 1.3% iD/g，注射后 1 小时）调查了。因此，选择[ 99m Tc]Tc-N 4 - L6 -CPCR4（称为[ 99m Tc]Tc-PentixaTec）进行首次人体应用。 [ 99m Tc]Tc-PentixaTec 具有良好的耐受性，在 SPECT 和平面成像中表现出良好的生物分布和剂量测定曲线（2.1–3.4 mSv/500 MBq）以及出色的肿瘤/背景比。
结论
连接子结构的氨基酸组成和 N 端99m Tc 标记策略（mas 3 vs HYNIC vs N 4 ）的连续优化使 [ 99m Tc]Tc-PentixaTec 成为一种新型、高度有前途的 CXCR4 靶向 SPECT临床应用剂。凭借其优异的CXCR4亲和力、高效的内化、CXCR4表达组织的高摄取、合适的清除/生物分布特性以及有利的人体剂量测定，其具有进一步临床应用的巨大潜力。
图形概要
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[99mTc]Tc-PentixaTec: development, extensive pre-clinical evaluation, and first human experience
Purpose
The clinical success non-invasive imaging of CXCR4 expression using [68 Ga]Ga-PentixaFor-PET warrants an expansion of the targeting concept towards conventional scintigraphy/SPECT with their lower cost and general availability. To this aim, we developed and comparatively evaluated a series of 99mTc-labeled cyclic pentapeptides based on the PentixaFor scaffold.
Methods
Six mas3-conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa3 linkers (L1–L6) as well as the corresponding HYNIC- and N4-analogs of L6-CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC50 and IC50inv) were carried out using Jurkat T cell lymphoma cells and [125I]FC-131 as radioligand. Internalization kinetics were investigated using hCXCR4-overexpressing Chem-1 cells. Biodistribution studies and small animal SPECT/CT imaging (1 h p.i.) were carried out using Jurkat xenograft bearing CB17/SCID mice. Based on the preclinical results, [99mTc]Tc-N4-L6-CPCR4 ([99mTc]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [99mTc]Tc-N4-L6-CPCR4 SPECT/planar imaging with individual dosimetry.
Results
Of the six mas3-conjugated peptides, mas3-L6-CPCR4 (mas3-dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC50 = 5.0 ± 1.3 nM). Conjugation with N4 (N4-L6-CPCR4) further improved hCXCR4 affinity to 0.6 ± 0.1 nM. [99mTc]Tc-N4-L6-CPCR4 also showed the most efficient internalization (97% of total cellular activity at 2 h) and the highest tumor accumulation (8.6 ± 1.3% iD/g, 1 h p.i.) of the compounds investigated. Therefore, [99mTc]Tc-N4-L6-CPCR4 (termed [99mTc]Tc-PentixaTec) was selected for first-in-human application. [99mTc]Tc-PentixaTec was well tolerated, exhibits a favorable biodistribution and dosimetry profile (2.1–3.4 mSv per 500 MBq) and excellent tumor/background ratios in SPECT and planar imaging.
Conclusion
The successive optimization of the amino acid composition of the linker structure and the N-terminal 99mTc-labeling strategies (mas3 vs HYNIC vs N4) has provided [99mTc]Tc-PentixaTec as a novel, highly promising CXCR4-targeted SPECT agent for clinical application. With its excellent CXCR4 affinity, efficient internalization, high uptake in CXCR4-expressing tissues, suitable clearance/biodistribution characteristics, and favorable human dosimetry, it holds great potential for further clinical use.
Graphical abstract
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