Fibroblast activation protein (FAP) is overexpressed on cancer-associated fibroblasts, making it an important target for cancer diagnosis and treatment, but limited tumor retention hinders late-stage diagnosis and radionuclide therapy. In this study, three albumin-bound FAP inhibitor (FAPI) radioligands, 68Ga/177Lu-DOTA-ALB-01, 68Ga/177Lu-DOTA-ALB-02, and 68Ga/177Lu-DOTA-ALB-03, were synthesized and evaluated for their in vitro stability, binding affinity, in vivo biodistribution, and tumor uptake using 68Ga and 177Lu labeling. All radioligands are stable in saline and plasma and exhibit high FAP-binding affinity. 177Lu-DOTA-ALB-02 has longer retention in circulation than 177Lu-FAPI-46 and other radioligands. Continuous tumor accumulation was observed during imaging with both 177Lu-DOTA-ALB-01 and 177Lu-DOTA-ALB-02. Notably, 177Lu-DOTA-ALB-02 had a significant tumor/nontarget ratio as indicated by biodistribution data. The outstanding tumor retention properties of 177Lu-DOTA-ALB-02 have been demonstrated in small-animal single-photon emission computed tomography imaging and biodistribution studies; therefore, it is considered the albumin-binding FAPI with the most favorable pharmacokinetic and imaging properties, worthy of further clinical investigation.

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©2025 The Authors; Published by the American Association for Cancer Research
2025
American Association for Cancer Research
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First page of Development and Application of Radioactive Ligands Targeting Fibroblasts with Albumin-Binding Sites<alt-title alt-title-type="short">Structure with Albumin Binder for FAP Targeting</alt-title>

Supplementary data

Table S1

Table S1. Stability data of 68Ga and 177Lu complexes of DOTA-LP, DOTA-ALB-01, DOTA-ALB-02, and DOTA-ALB-03 in human serum (HS)and PBS buffer, expressed as the percentage of stable complexes (percentage of labeled compound to free activity found at a given time point), with the corresponding analytical conditions detailed in the main text.

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Table S2

Table S2.Binding affinity and IC50 values for FAPI-46 and FAPI derivatives toward hFAP

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Table S3

Table S3.Organ biodistribution of [177Lu]Lu-FAPI-46 in U87MG tumor model as %ID/g tissue. Data are expressed as mean ± SD (n=3-5).

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Table S4

Table S4.Organ biodistribution of [177Lu]Lu-DOTA-LP in U87MG tumor model as %ID/g tissue. Data are expressed as mean ± SD (n = 3–5).

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Table S5

Table S5.Organ biodistribution of [177Lu]Lu-DOTA-ALB-01 in U87MG tumor model as %ID/g tissue. Data are expressed as mean ± SD (n=3-5).

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Table S6

Table S6.Organ biodistribution of [177Lu]Lu-DOTA-ALB-02 in U87MG tumor model as %ID/g tissue. Data are expressed as mean ± SD (n=3-5).

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Table S7

Table S7.Organ biodistribution of [177Lu]Lu-DOTA-ALB-03 in U87MG tumor model as %ID/g tissue. Data are expressed as mean ± SD (n=3-5).

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Figure S1

Figure S1.ESI-MS spectrum and HPLC analysis of DOTA-LP. See main text for corresponding analytical conditions.

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Figure S2

Figure S2. ESI-MS spectrum and HPLC analysis of DOTA-ALB-01. See main text for corresponding analytical conditions.

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Figure S3

Figure S3. ESI-MS spectrum and HPLC analysis of DOTA-ALB-02. See main text for corresponding analytical conditions.

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Figure S4

Figure S4. ESI-MS spectrum and HPLC analysis of DOTA-ALB-03. See main text for corresponding analytical conditions.

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Figure S5

Figure S5. Synthesis of DOTA-LP, DOTA-ALB-01, and DOTA-ALB-02. The corresponding synthesis method is described in the main text.

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Figure S6

Figure S6. Synthesis of DOTA-ALB-03.The corresponding synthesis method is described in the main text.

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Figure S7

Figure S7. Radio-HPLC analysis of 68Ga-DOTA-LP,68Ga-DOTA-ALB-01,68Ga-DOTA-ALB-02, and 68Ga-DOTA-ALB-03.

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Figure S8

Figure S8. Radio-HPLC analysis of 177Lu-DOTA-LP, 177Lu-DOTA-ALB-01, 177Lu-DOTA-ALB-02, and 177Lu-DOTA-ALB-03.

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Figure S9

Figure S9. (A) Radio-HPLC analysis of 68Ga-labeled compounds’ stabilities in saline and serum for 2 h at 37 °C. (B) Radio-HPLC analysis of 177Lu-labeled ligands’ stabilities in saline and serum for 24 h at 37 °C.

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Figure S10

Figure S10. Binding characteristics of radio ligands with human serum albumin.

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Figure S11

Figure S11. in vivo blocking and specificity of 68Ga-DOTA-LP (A), 68Ga-DOTA-ALB-01 (B),68Ga-DOTA-ALB-02 (C), and68Ga-DOTA-ALB-03 at 1 h p.i.

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Figure S12

Figure S12. Micro-PET/CT imaging of 68Ga-labelled ligands in U87MG tumor-bearing mice at 0.5,1, and 2 p.i. (tumors are marked with white arrows).

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Figure S13

Figure S13. Micro-SPECT/CT imaging of 177Lu-FAPI-46, 177Lu-DOTA-LP, and 177Lu-DOTA-ALB-03 in U87MG tumor-bearing mice at 1, 4 h (tumors are marked with white arrows).

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