FAPI Dimerization for Theranostic Applications: Molecular Design, Preclinical Validation, and Clinical Translation

Abstract

Fibroblast activation protein (FAP), a serine protease overexpressed in cancer-associated fibroblasts of >90% epithelial malignancies, has emerged as a highly promising pan-cancer target for theranostic radiopharmaceuticals. However, the rapid clearance of monomeric FAP inhibitors (FAPIs) limits their therapeutic efficacy despite excellent diagnostic performance. Multimerization strategies-particularly dimeric and heterodimeric constructs-have been developed to overcome this limitation through enhanced binding avidity and prolonged tumor retention. This review provides a comprehensive analysis of recent advances in FAPI-based multimers, focusing on their molecular design, chemical synthesis, preclinical evaluation, and early clinical applications. We examine the role of linker chemistry (length, flexibility, cleavability) and chelator selection in optimizing pharmacokinetics and tumor-to-background contrast. Key examples include homodimers (e.g., DOTA-2P­(FAPI)₂, BiOncoFAP) that leverage the polyvalency effect, and heterodimers (e.g., FAPI-RGD, PSFA-01) that enable dual-targeting of FAP and complementary receptors such as integrin α_vβ₃ or PSMA. These probes show superior tumor uptake and retention in preclinical models and have demonstrated enhanced diagnostic sensitivity and therapeutic potential in clinical trials across multiple cancer types. Beyond oncology, emerging applications in fibrotic and inflammatory diseases-such as rheumatoid arthritis and interstitial lung disease-highlight the versatile utility of FAPI multimers. While challenges including renal uptake, synthetic complexity, and cost remain, ongoing innovations in chemical design and combination therapies position these agents as transformative tools in precision theranostics, bridging high-contrast imaging with effective radioligand therapy for personalized patient management.

1. Introduction

The tumor microenvironment (TME) represents a complex and dynamic ecosystem comprising cancer cells, immune cells, and extracellular matrix, which collectively support tumor progression and therapeutic resistance. , This protective niche is not a passive bystander but an active participant in tumorigenesis, facilitating cancer cell proliferation, invasion, metastasis, and resistance to therapy. Unlike genetically unstable tumor cells that rapidly evolve under therapeutic pressure, stromal elements like CAFs exhibit relative genetic stability and phenotypic consistency, making them attractive and potentially more durable therapeutic targets. Activated CAFs secrete a plethora of growth factors, cytokines, and proteases that promote angiogenesis, ECM remodeling, local immunosuppression, and ultimately, tumor progression. ,

A key functional and phenotypic marker of activated CAFs is fibroblast activation protein (FAP), a membrane-bound serine protease with dipeptidyl peptidase and collagenase activities. FAP is directly involved in CAF-mediated ECM degradation, growth factor signaling, and immune modulation within the TME, thereby driving cancer aggressiveness. Crucially, FAP expression is highly selective: it is significantly overexpressed in the stroma of >90% of epithelial carcinomas (including pancreatic, breast, lung, and colorectal cancers) while maintaining negligible expression in most healthy adult tissues. This stark expression dichotomy renders FAP an exceptionally attractive pan-cancer target for molecular imaging and targeted radionuclide therapy.

Capitalizing on this profile, first generation monomeric (FAPI)-based radiotracers (e.g., FAPI-04, FAPI-46) have demonstrated excellent performance in clinical PET imaging, offering high-contrast visualization of primary and metastatic lesions across numerous cancer type. However, their translation from diagnostic tools to effective radioligand therapy (RLT) agents has been fundamentally hampered by short intratumoral retention, creating a pharmacokinetic mismatch with the long half-lives of therapeutic radionuclides (e.g., ¹⁷⁷Lu, 6.7 days; ²²⁵Ac, 9.9 days). This mismatch results in insufficient radiation dose delivery to tumors and compromised therapeutic efficacy.

To address these critical limitations, innovative multimerization strategies have been developed. By conjugating two or more FAPI pharmacophores onto a single molecular scaffold via optimized chemical linkers, these multivalent constructs leverage the “polyvalency effect”. This effect enhances functional binding avidity through simultaneous engagement of multiple FAP receptors, promotes receptor clustering and internalization, and dramatically extends tumor retention time-often from hours to several days. Furthermore, heterodimeric probes, which combine a FAPI ligand with a ligand targeting a distinct tumor-associated antigen (e.g., integrin α_vβ₃ or prostate-specific membrane antigen PSMA), offer a powerful strategy to overcome intratumoral and intertumoral heterogeneity. Such probes can improve diagnostic sensitivity by capturing multiple tumor compartments and expand therapeutic coverage. ,

Fibroblast activation protein (FAP) is currently a primary target in oncology. Recently, several reviews have highlighted the rapid development of FAP-targeted radiopharmaceuticals. For example, Wu et al. systematically summarized the evolution of FAPI tracers from drug design to clinical translation, with a specific focus on structural modification strategies. Unlike previous comprehensive reviews, this review aims to provide a comprehensive and critical examination of the latest advances in FAPI-based multimers, with a focused analysis on homodimers and heterodimers. We will systematically explore: (1) the molecular design rationale and synthetic chemistry; (2) the critical roles of linker and chelator optimization; (3) preclinical evaluation of pharmacokinetics, imaging, and therapy; (4) emerging clinical translation and applications in oncology and beyond; and (5) future perspectives and unresolved challenges. By synthesizing this rapidly evolving field, we highlight the transformative potential of FAPI multimers to advance precision radionuclide theranostics.

2. Molecular Design and Chemical Synthesis of FAPI Dimers

Design Rationale: The Polyvalency Effect

The polyvalency effect, where multiple copies of a targeting ligand on a single scaffold enhance effective receptor engagement, has been successfully exploited in other systems like PSMA. , In the development of FAP-targeted dimeric probes such as DOTA-2P­(FAPI)₂, based on the FAPI-46 scaffold, consistently demonstrated superior tumor uptake and retention compared to monomers. The foundational principle behind FAPI multimerization is the polyvalency effect, a well-established phenomenon in molecular recognition where multiple copies of a ligand presented on a single scaffold cooperatively bind to multiple adjacent receptors, resulting in an apparent binding affinity (avidity) that is significantly greater than the sum of its individual binding events. This strategy has been successfully leveraged in other targeted radiopharmaceutical systems, most notably in PSMA-targeting agents (e.g., PSMA-617 dimers and trimers). Applied to FAP targeting, dimeric probes such as DOTA-2P­(FAPI)₂, constructed from two FAPI-46 pharmacophores linked via a triethylene glycol (PEG3) spacer, consistently demonstrate superior tumor targeting compared to the FAPI-46 monomer. In a hepatocellular carcinoma patient-derived xenograft (PDX) model, ⁶⁸Ga-DOTA-2P­(FAPI)₂ (Figure ) showed significantly higher and more sustained tumor accumulation than ⁶⁸Ga-FAPI-46. Similarly, the high-affinity dimeric derivative, ¹⁷⁷Lu-BiOncoFAP, achieved markedly higher tumor uptake (∼20%ID/g vs ∼ 4%ID/g for its monomer) at 24 h postinjection while maintaining favorable tumor-to-organ ratios, underscoring the therapeutic promise of prolonged retention.

1.

Representative chemical structures of FAPI multimers. (A) Structure of the heterodimer PSFA-01, targeting both PSMA and FAP. (B) Structure of the homodimer DOTA-2P­(FAPI)₂, based on the FAPI-46 pharmacophore linked via a PEG₃ spacer and chelated by DOTA.

The valency-activity relationship has been further explored with higher-order multimers. For instance, a FAPI tetramer, DOTA-4P­(FAPI)₄ was developed and compared against its dimeric and monomeric analogs. In HT-1080-FAP tumor models, the ¹⁷⁷Lu-labeled tetramer exhibited the highest tumor uptake (21.4 ± 1.7%ID/g at 24 h) and the longest retention, significantly higher than the dimer (17.1 ± 3.9%ID/g) and monomer (3.4 ± 0.7%ID/g). Consequently, the tetramer induced the most pronounced tumor growth inhibition, confirming that increasing valency can further enhance tumor accumulation and therapeutic potential, albeit with potential trade-offs in synthetic complexity and pharmacokinetics.

Linker Chemistry and Optimization

The linker, which covalently connects the chelator/radionuclide complex to the targeting pharmacophores, is not a mere structural tether but a critical “molecular tuner” that profoundly influences spatial conformation, target affinity, metabolic stability, pharmacokinetics (PK), and biodistribution.

Length and Flexibility

Linker length acts as a “spacer arm,” determining the optimal distance between pharmacophores for simultaneous bivalent binding to FAP dimers or clusters on the cell membrane. Studies using monodispersed PEGs (PEG₁₂, PEG₂₄, PEG₄₈) have demonstrated that increasing linker length can modulate clearance pathways-often reducing kidney uptake and prolonging circulation time, which in turn can enhance tumor accumulation through the enhanced permeability and retention (EPR) effect. However, optimization is not linear; excessively long or flexible linkers may increase entropy and reduce effective local concentration, impairing binding efficiency. Conversely, shorter “mini-PEG” or aliphatic linkers can minimize steric hindrance, improve radiolabeling efficiency, and maintain high target affinity. In summary,linker length plays a pivotal role in the probe’s ability to precisely match the target’s spatial configuration and improve in vivo pharmacokinetics.

Chemical Nature and Cleavability

Linkers are broadly categorized as noncleavable or cleavable. Noncleavable linkers (e.g., stable amide, triazole, or PEG chains) provide structural integrity and predictable, stable pharmacokinetics, ensuring the radionuclide remains attached to the targeting moiety until cellular internalization and lysosomal degradation. Cleavable linkers designed as “smart” switches, that exploit physiological differences between the TME and normal tissues. Examples include:

Enzyme-responsive linkers: Substrates for proteases overexpressed in the TME (e.g., matrix metalloproteinases, cathepsins). Notably, linkers susceptible to cleavage by FAP’s own protease activity upon binding represent an elegant “prodrug” strategy, potentially releasing a more potent payload or modulating clearance.

Redox-responsive linkers: Cleavable by high intracellular glutathione (GSH) concentrations in tumor cells (e.g., disulfide bonds).

Hydrophilicity/Hydrophobicity

The overall hydrophilicity of the probe, heavily influenced by the linker, dictates its excretion pathway and potential organ toxicity. Hydrophilic probes (lower logP), often achieved using PEG or sugar-based linkers, favor renal excretion, which is generally desirable but can lead to high kidney radiation doses-a primary concern for RLT. , Lipophilic probes undergo hepatobiliary excretion, which may increase radiation exposure to the liver, intestines, and colon, potentially causing gastrointestinal toxicity and complicating abdominal imaging. Therefore, linker design often aims to fine-tune hydrophilicity to balance rapid blood clearance with acceptable organ uptake. The hydrophilicity of the linkers is summarized in Table .

1. Influence of Linker and Chelator Modifications on the Properties of Selected FAPI ,

Chelator Selection and Radiolabeling Strategies

Stable complexation of the radiometal is paramount for safety and efficacy. Direct radioiodination (e.g., ¹³¹I) can lead to in vivo dehalogenation and premature release of radioactivity, causing off-target toxicity. The predominant strategy is indirect labeling using macrocyclic chelators that form thermodynamically stable and kinetically inert complexes with radiometals. Common Chelators: DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid): The workhorse chelator, versatile for a wide range of diagnostic (⁶⁸Ga³⁺, ⁶⁴Cu²⁺) and therapeutic (¹⁷⁷Lu³⁺, ⁹⁰Y³⁺, ²²⁵Ac³⁺) radionuclides. NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and NODAGA: Offer faster and milder labeling conditions for ⁶⁸Ga³⁺, often yielding higher specific activity. DOTAGA and DATA derivatives: Provide alternative coordination geometries and stability profiles.

The choice of chelator is often dictated by the radionuclide. Innovative designs incorporate multiple distinct chelators on a single scaffold (e.g., a SiFA moiety for ¹⁸F-aluminum fluoride labeling combined with DOTA for radiometals), enabling flexible “mix-and-match” labeling with different isotopes for diagnostic or therapeutic purposes from the same precursor molecule.

Radionuclide Pairing for Theranostics

The concept of true “theranostics” relies on using chemically identical or highly similar vectors paired with matched radionuclide pairs.

Diagnostic: ⁶⁸Ga (T _1/2 = 68 min, β⁺ emitter for PET), ¹⁸F (T _1/2 = 110 min), ⁶⁴Cu (T _1/2 = 12.7 h).

Therapeutic: ¹⁷⁷Lu (T _1/2 = 6.7 d, medium-energy β^– emitter), ⁹⁰Y (T _1/2 = 2.7 d, high-energy β^– emitter), ²²⁵Ac (T _1/2= 9.9 d, α emitter).

The prolonged tumor retention achieved by dimerization (biological T _1/2 extended to days) creates a much better temporal match with the physical half-life of therapeutic nuclides like¹⁷⁷Lu, enabling effective radiation dose delivery and deepening the integration of diagnosis and therapy.

3. Common Structural Types of FAPI Multimers

Homodimers

Early and extensively studied multimers are homodimers, featuring two identical FAPI pharmacophores connected via a linker to a central chelator. This symmetric design capitalizes on the polyvalency effect for FAP. Benefits include improved plasma stability, enhanced tumor binding avidity, increased tumor-to-background ratios, and elevated diagnostic sensitivity. A prime example is DOTA-2P­(FAPI)₂, which has shown prolonged tumor retention and higher uptake compared to FAPI-46 in multiple PDX models and in pilot clinical studies. The overview of the studies on homodimers is presented in Table .

2. Summary of Clinically Relevant Dimeric FAPI Constructs.

Probe Name	Linker	Chelator	Affinity vs Monomer	Tumor Retention Compared to Monomers (%ID/g)	Advantages Compared to Monomers	Clinical Application	ref.
ND-bisFAPI	Asp-Lys	NOTA/DOTA	Affinity similar IC5 0= 2.0 (8-fold potency vs monomer)	Significant prolongation of retention time (4 h: 12.3 ± 4.2)	Achieves comparable therapy efficacy at 50% monomer dose; high tumor absorbed dose.	Tumor imaging and therapy
DOTAGA.Glu.(FAPi) 2	Glutamic acid	DOTAGA	-	Significant prolongation of retention time (96 h: 2.2 ± 0.2)	High hydrophilicity reduces hepatobiliary excretion; compatible with α-therapy (225Ac).	Tumor imaging and therapy
DOTAGA.(SA.FAPi) 2	Squaric Acid	DOTAGA	Affinity similar IC5 0≈ 1–2 nM	Significant prolongation of retention time (Retention up to 96 h)	validated safety and prolonged retention in metastatic breast/thyroid cancers.	Tumor imaging and therapy	,
BiOncoFAP	-	-	Affinity stronger IC5 0= 0.17 nM	Significant prolongation of retention time (48h: 16.46)	Highest reported affinity/initial uptake with >50% retention at 48 h, strong single high-dose RLT potential.	Tumor imaging and therapy
FAPI-46-F1D	-	DOTA	Affinity stronger	Significant prolongation of retention time	Doubles the tumor radiation dose compared to the monomer	Tumor imaging and therapy
Gd-DOTA-Suc-Lys-(FAPI) 2	Suc-Lys	DOTA(Gd)	-	Significant prolongationof retention time (0–30 min: Significant contrast)	Dimerization significantly improves this MRI probe’s targeting and imaging of FAP-positive tumors.	Tumor imaging
[ 68 Ga]Ga-LNC1013	-	DOTA	Affinity similar IC50= 7.15 nM	Significant prolongationof retention time (prolonged high uptake)	First-in-human study confirmed superior tumor accumulation vs monomeric analogs.	Tumor imaging
DOTA-2P(FAPI) 2	PEG3	DOTA	IC5 0 = 3.68 ± 1.82 nM (Monomer 2.06 ± 1.84 nM)	Significant prolongationof retention time	Long retention, synergistic PD-L1	Tumor imaging and therapy	, ,
DOTA-Suc-Lys-(FAPI-04) 2	Suc-Lys	DOTA	Affinity similar	Significant prolongationof retention time (Uptake 2-fold vs monomer, No significant decrease even at 3 h)	Long-lastingretention, tumor suppression and significant efficacy.	Tumor imaging and therapy
FAPI-JNU	Contains Glutamic acid	DOTA	picomolar affinity for FAP	Significant prolongationof retention time, Higher tumor uptake observed compared to 68Ga-FAPI-46	High affinity, excellent imaging results and effectively inhibit tumor growth	Tumor imaging and therapy

Heterodimers

This innovative class conjugate a FAPI ligand to a ligand targeting a different, complementary receptor. This strategy retains the benefits of multivalency while addressing tumor heterogeneity by targeting two distinct biological pathways-often the stromal FAP component and a tumor-cell-specific antigen. This can significantly improve overall diagnostic sensitivity and therapeutic coverage. Prominent examples include:

FAPI-RGD

Targets FAP on CAFs and integrin α_vβ₃ on tumor neovascularure and some cancer cells. In preclinical Panc02 models, ⁶⁸Ga-FAPI-RGD showed significantly higher tumor uptake (5.95 ± 0.43%ID/g at 1 h) than either ⁶⁸Ga-RGDfK (3.51 ± 0.47%ID/g) or ⁶⁸Ga-FAPI-02 (0.97 ± 0.09%ID/g) alone. Its clinical utility across multiple cancers is detailed in later sections.

FAPI-PSMA (e.g.,⁶⁸Ga-PSFA-01)

Targets PSMA on prostate cancer cells and FAP on the surrounding reactive stroma. This probe has demonstrated a higher detection rate for metastatic prostate cancer lesions, particularly identifying PSMA-negative but FAP-positive lesions that would be missed by single-target PSMA tracers, thereby providing a more comprehensive disease assessment. ,

SSTR2-FAP (e.g.,⁶⁸Ga-FAPI-LM3)

Targets somatostatin receptor subtype 2 (SSTR2) on neuroendocrine tumor cells and FAP on associated fibroblasts. In patients with nasopharyngeal carcinoma (NPC), this heterodimer demonstrated superior lesion detection and higher uptake compared to ¹⁸F-FDG and ⁶⁸Ga-FAPI-46, improving staging accuracy. −

4. Preclinical Evaluation

In Vitro Binding Studies

Cell-based assays consistently confirm the enhanced avidity of dimeric probes. Uptake of ⁶⁸Ga-DOTA-2P­(FAPI)₂ in FAP-expressing CAFs was approximately twice that of the monomeric ⁶⁸Ga-FAPI-46. Competitive binding assays, while sometimes showing similar intrinsic affinity (IC₅₀) at the single-binding-site level, demonstrate the superior functional avidity of dimers in cellular contexts. The dual-targeting ⁶⁸Ga-PSFA-01 exhibits high affinity for both FAP and PSMA proteins, with K _i values in the subnanomolar to low nanomolar range (0.14–1.02 nM). Crucially, efflux assays reveal intracellular consequences of multivalency: ¹⁷⁷Lu-BiOncoFAP displayed a significantly longer intracellular half-life (36 h) compared to its monomeric form (18 h), directly linking enhanced retention to multivalent binding and internalization.

In Vivo Biodistribution and Imaging

Ex vivo biodistribution studies across various xenograft models uniformly show that dimerization significantly increase tumor uptake and retention times. In the A431 xenograft, uptake of ⁶⁸Ga-(FAPI-04)₂ reached 6.51 ± 0.98%ID/g early postinjection (∼6 times the monomer) showed significant retention even at 72 h. Similarly prolonged retention was observed for ¹⁷⁷Lu-BiOncoFAP.

However, optimization is critical as modifications can alter nontarget organ pharmacokinetics. Increased molecular weight and hydrophilicity from dimerization and specific linkers can lead to slower blood clearance and variable effects on kidney and liver uptake. For instance, some dimers show elevated and persistent kidney activity, which is a primary concern for RLT due to potential nephrotoxicity. Conversely, probes like ⁶⁸Ga-FAPI-LM3demonstrated optimized clearance profiles with high tumor-to-background contrast. These findings underscore that future research must focus on fine-tuning chemical structures (e.g., linker polarity, charge) to optimize the balance between high tumor retention and low healthy tissue exposure.

Imaging studies visually confirm these advantages. In the study of the dual-targeting heterodimeric radiotracer Micro-PET/CT imaging of ⁶⁸Ga-FAPI-RGD in Panc02 tumor-bearing mice showed superior tumor visualization and higher tumor-to-background ratios at all time points compared to the single-target monomers ⁸Ga-RGDfK and ⁶⁸Ga-FAPI-02 (Figure ).

2.

Preclinical PET imaging with ⁶⁸Ga-DOTA-2P­(FAPI)₂ (A), ⁶⁸Ga-FAPI-LM3 (B), ⁶⁸Ga-TATE-46 (C), ⁶⁸Ga-FAPI-RGD (D). Representative maximum intensity projection (MIP) PET images of tumor-bearing mice at 0.5, 1, 2, and 4 h postinjection of demeric probes and monomeric probes. (A) Reproduced with permission from ref . Copyright 2022 Society of Nuclear Medicine and Molecular Imaging. (B) Reproduced with permission from ref . Copyright 2024 Society of Nuclear Medicine and Molecular Imaging. (C) Reproduced with permission from ref . Copyright 2024 Liu et al. (D) Reproduced with permission from ref . Copyright 2022 Zang et al.

Therapeutic Efficacy Studies

The extended tumor retention of FAPI dimers directly translates to enhanced therapeutic potential in preclinical RLT models. Galbiati et al. demonstrated that a single dose of ¹⁷⁷Lu-BiOncoFAP significantly inhibited tumor growth in a dose-dependent manner in HT-1080. FAP xenografts, validating the dimer’s therapeutic efficacy. Beyond standalone RLT, combination strategies are emerging. Chen et al. leveraged the ability of ¹⁷⁷Lu-DOTA-2P­(FAPI)₂ to induce immunogenic cell death and upregulate PD-L1 in a CT26-FAP colorectal cancer model.Combining this RLT with an anti-PD-L1 antibody (αPD-L1) yielded a potent synergistic antitumor effect, leading to tumor elimination and significantly prolonged survival in mice. This highlights the promising avenue of combining FAPI-targeted RLT with immunotherapy.

5. Clinical Translation and Applications

Diagnostic Applications in Oncology

Novel FAPI dimeric and heterodimeric probes are rapidly transitioning from bench to bedside, demonstrating superior imaging performance in clinical PET/CT (Figure ). An overview of the preclinical and clinical studies on heterodimers is provided in Table .

3.

Clinical PET/CT imaging examples of FAPI heterodimers in various tumors. Maximum intensity projection (MIP) and fused axial PET/CT images demonstrating high uptake of heterodimeric probes in various malignancies: (A) ⁶⁸Ga-FAPI-JNU in a patient with pancreatic cancer. Reproduced with permission from ref . Copyright 2025 American Chemical Society. (B, C) ⁶⁸Ga-FAPI-RGD in patients with nasopharyngeal carcinoma and lung adenocarcinoma metastases.Reproduced with permission from ref . Copyright 2023 Society of Nuclear Medicine and Molecular Imaging. (D) ¹⁸F-AlF-LNC1007 in a patient with renal cell carcinoma. Reproduced with permission from ref . Copyright 2025 Gao et al. (E) ⁶⁸Ga-PSFA-01 in a patient with metastatic prostate cancer. Reproduced with permission from ref . Copyright 2023 Wang et al.

3. Characteristics and Clinical Value of FAPI-Based Heterodimer Radiotracer.

Radiotracer	Dual Targets	Study Design	Indication/Tumor Type	Comparative Efficacy vs 18F-FDG or Monomeric Tracers	Clinical & Translational Value	Study (ref.)
68 Ga-FAPI-RGD	FAP + Integrin αvβ3	Clinical	Pan-cancer(Panc02 model +6 patients)	SUVmax: Tumor SUVmax comparable to 18F-FDG (no significant difference)	Established favorable pharmacokineticsfor FAPI-RGD in humans; validated high Tumor-to-Background Ratios (TBR).	Zang et al. 2022
				Superior to Monomers: Significantly improved tumor uptake/retention and cleaner background in preclinical models.
			Lung neoplasms (Primarily NSCLC)	Tumor SUVmax comparable to 18F-FDG(SUVmax, 6.9 ± 5.3 vs 5.3 ± 5.4, p < 0.001). Outperforms 18F-FDG: Higher detection rate (91.4% vs 77.1%,P < 0.05), SUVmax (P < 0.001), and TBR. More accurate mediastinal LN staging.	Dual-targeting yields higher sensitivity for primary and metastatic lesions	Wang et al. 2023
			Various cancers (NPC, breast, esophageal, lung, pancreatic)	Primary tumors: SUVmax 18.0 (vs 9.1 for FDG). LN metastases: SUVmax 12.1 (vs 6.1).Bone metastases: SUVmax 16.2. Superior to 18F-FDG: Significantly higher SUVmax and TBR across primary and metastatic lesions (e.g., bone detection 100% vs 80%).	Exceptional tumor-to-background ratio (TBR); substantially improved detection of lymph node and bone metastases.	Zhao et al. 2023
				Superior to [68Ga]Ga-FAPI-46 in metastatic sites.
18 F-FAPI-42-RGD		Preclinical	Gastric cancer(MKN-45, N87–18.2, PDX)	Across the tumor models evaluated, tumor uptake was significantly enhanced at all assessed time points compared with the monomer. Superior to Monomer: 2–3 fold higher tumor uptake compared to 18F-FAPI-42 alone; clearer background.	Potential for Clinical Translation in Gastric Cancer (GC) Imaging and Diagnosis	Zhao et al. 2025
[ 18 F]AlF-LNC1007		Clinical	Renal cell carcinoma (RCC)	Primary Tumor SUVmax: 8.31 (vs 4.82 for FDG).	This dual-tracer demonstrated significantly higher diagnostic efficacy and superior tumor uptake compared to both 18F-FDG and single-target FAPI tracers.	Gao et al. 2025
([ 18 F]AlF-NOTA-FAPI-RGD)				Superior to 18F-FDG: Significantly higher sensitivity (100% vs 85%) and TBR (P < 0.001). Superior to [18F]AlF-FAPI-04: Higher uptake and detection accuracy (98% vs 90%).
18 F-FAPI-RGD		Clinical	Connective tissue disease-associated interstitial lung disease (CTD-ILD)	Bilateral lung SUVmax significantly higher in CTD-ILD vs non-ILD controls (P < 0.001).	High lung SUVmax correlates strongly with disease activity, inflammatory markers, and pulmonary function decline.	Liu et al. 2025
[ 18 F]AlF-FAPI-RGD		Clinical	Rheumatoid arthritis (RA)	Joint Detection Rate: 82.4% (PET) vs 68.4% (Clinical assessment).	Objective molecular assessment of inflammation/fibrosis activity; potential for monitoring therapeutic response (responders showed significantly decreased SUVmax).	Wang et al. 2024
				N/A (vs Clinical): Detected subclinical synovitis and interstitial lung disease (ILD) lesions missed by standard physical exams.
64 Cu-FP-L1/ 64 Cu-FP-L2	FAP + PSMA	Preclinical	Prostate cancer, glioblastoma, pancreatic cancer	High uptakeobserved in dual-positive (FAP+/PSMA+) models.	Prolonged tumor retention profile supports future translation to therapeutic radionuclides (e.g., 177Lu).	Boinapally et al. 2022
				Superior to Monomer: Superior uptakein tumors coexpressing both targets compared to single-target controls.
68 Ga-PSFA-01		Clinical	Prostate cancer (mCRPC)	Demonstrated superior performance in both patient-based and lesion-based analyses, with a higher detection rate compared to the monomeric counterpart.	Addressing heterogeneity: Effective for patients with low or heterogeneous PSMA expression.	Li et al. 2025
68 Ga-AV01084		Preclinical	Prostate Cancer(LNCaP, HEK-FAP)	The tumor uptake values from the dual-targeting tracers were still lower than those obtained from the monospecific tracers.	Demonstrated specific binding feasibility but highlighted the challenge of linker optimization in dual-targeting tracer design.	Verena et al. 2024
68 Ga-AV01088
[ 18 F]AlF-PSMA-FAPI-01/02		Preclinical	Lung (A549-FAP) & Prostate (22Rv1)	Tumor uptake: Higher than monomers superior to monomers: Significantly higher uptake than [18F]FAPI-42 and [18F]PSMA-BCH in respective positive models (P < 0.05).	Successfully demonstrated that 18F-labeled heterodimers can achieve high affinity and specificity for both targets with favorable pharmacokinetics.	Hu et al. 2022
68 Ga-FAPI-LM3	FAP + SSTR2	Preclinical	Pulmonary Fibrosis(Bleomycin model)	Lung uptake: 1.90%ID/g (vs 0.78 for FAPI-46 and 0.45 for DOTA-LM3) at 60 min. Superior to monomers: Significantly higher accumulation in fibrotic lesions compared to single-target tracers.	Demonstrated potential for detecting early stage pulmonary fibrosis (Day 7) where monomers showed only low-to-medium uptake.	Chen et al. 2024
		Clinical	Nasopharyngeal Carcinoma (NPC)	Primary tumor SUVmax:13.8 (vs 9.3 for FDG).	Targets both tumor cells (SSTR2) and stroma (FAP), resulting in superior retention and sensitivity for initial staging and recurrence.	Zhao et al. 2024
				Superior to 18F-FDG: Detected additional metastases missed by FDG.
				Superior to FAPI-46: Higher uptake and retention.
68 Ga-TATE-46		Preclinical	Neuroendocrine Tumor (NCI-H727)	Tumor uptake: Significantly higher than monomers (P < 0.001).	Improves tumor uptake, extends tumor retention, and enhances pharmacokinetics.	Liu et al. 2024
				Superior to monomers: Higher accumulation than 6 8Ga-FAPI-46 and 68Ga-DOTA-TATE individually.

Pan-Cancer Imaging with FAPI-RGD

In a clinical evaluation involving 22 patients with various cancer (nasopharyngeal carcinoma, breast cancer, esophageal cancer, lung cancer, pancreatic cancer, and ovarian cancer) compared ⁶⁸Ga-FAPI-RGD with ¹⁸F-FDG. The heterodimer showed significantly higher SUVmax in primary tumors (18.0 vs 9.1) and metastases, and higher tumor-to-background ratios (TBR). Critically, it detected lesions negative on ⁶⁸Ga-FAPI-46 PET imaging but positive for integrin α_vβ₃, proving the dual-targeting strategy overcomes false negatives from single-target heterogeneity.

Lung Cancer

In 51 patients with suspected lung neoplasms, ⁶⁸Ga-FAPI-RGD PET/CT showed a higher detection rate for primary lesions (91.4% vs 77.1%), tumor-to-background ratios (TBR, 6.9 ± 5.3 vs 5.3 ± 5.4) and higher SUVmax and superior accuracy for mediastinal lymph node stagingcompared to ¹⁸F-FDG (99.7% vs 90.9%). Additionally, the uptake value of ⁶⁸Ga-FAPI-RGD was positively correlated with the expression levels of FAP and integrin α_vβ₃.

Renal Cell Carcinoma (RCC)

Due to the low glucolysis, ¹⁸F-FDG performs poorly in RCC. The heterodimer ¹⁸F-AlF-LNC1007 (i.e., ¹⁸F-AlF-NOTA-FAPI-RGD) demonstrated significantly higher detection rates (91% vs 76%; 94% vs 34%), sensitivity (100% vs 85%; 94% vs 29%), and SUVmax values in both primary lesions and metastases RCC lesions compared to ¹⁸F-FDG and the monomer ¹⁸F-AlF-NOTA-FAPI-04.

Applications in Nononcologic Diseases

The biology of FAP (fibrosis) and integrin α_vβ₃ (angiogenesis/inflammation) extends the utility of these tracers beyond oncology.

Rheumatoid Arthritis (RA)

In 28 patients with active RA, ¹⁸F-AlF-FAPI-RGD PET/CT visualized synovitis with high sensitivity. Its quantitative parameters significantly correlate with clinical disease activity scores and detected subclinical lesions missed by conventional methods, showing great potential for therapy monitoring.

Interstitial Lung Disease (ILD)

In 52 patients with connective tissue disease-associated CTD-ILD, ¹⁸F-FAPI-RGD PET/CT successfully differentiated patients from controls. Lung SUVmax showing excellent predictive value for ILD and correlated with pulmonary function decline, offering a potential tool for early diagnosis and activity assessment.

6. Head-to-Head Comparison with FAPI Monomers

Direct clinical comparisons solidify the advantages of multimers. In prostate cancer, ⁶⁸Ga-PSFA-01 detected more lesions than ⁶⁸Ga-PSMA-11 and ⁶⁸Ga-FAPI-04 in the same patients, effectively identifying PSMA-low/FAP-high metastases. Similarly, in nasopharyngeal carcinoma, ⁶⁸Ga-FAPI-LM3 visualized more lesions with higher uptake intensity than ⁶⁸Ga-FAPI-46.

7. Theranostics: Integrating Diagnosis and Therapy

Integration of Theranostics in Dimeric FAPI

The true power of FAPI multimers is realized in the theranostic paradigm. This approach uses chemically identical or highly similar probes for both diagnostic imaging and subsequent targeted radiotherapy.

Diagnostic Phase: A patient undergoes PET/CT with a diagnostic radionuclide (e.g., ⁶⁸Ga-labeled FAPI dimer). The scan provides high-quality, whole-body mapping of all FAP-positive lesions, enabling precise staging and patient selection for therapy.

Therapeutic Phase: If the diagnostic scan confirms high and specific tumor uptake, the same or an analogous FAPI dimer construct, labeled with a therapeutic radionuclide (e.g., ¹⁷⁷Lu), is administered. Due to the identical targeting vector, the therapeutic agent biodistributes to the exact same lesions “seen” on the diagnostic scan.

Personalized Treatment: This ensures precise, tumor-focused delivery of cytotoxic radiation while sparing healthy tissues, epitomizing personalized medicine. The prolonged retention of dimers ensures sufficient radiation dose delivery over the nuclide’s half-life.

Therapeutic Efficacy: The Role of Dimerization

The development of FAPI probes has long focused on extending tumor retention. By introducing multivalency and chemical modificationssuch as the incorporation of SuFEx warheads-probe retention can be significantly prolonged, thereby substantially enhancing the therapeutic efficacy of FAPI probes.

Preclinical studies with probes like ¹⁷⁷Lu-DOTA-2P­(FAPI)₂ and ¹⁷⁷Lu-(FAPI-04)₂ have successfully validated this “see what you treat” strategy, showing significant tumor growth inhibition. Early clinical applications, in metastatic breast cancer with ¹⁷⁷Lu-DOTAGA.(SA.FAPi)₂. Compared to its monomeric counterpart, ¹⁷⁷Lu-DOTAGA.(SA.FAPi)₂ demonstrated significantly prolonged retention, resulting in a substantial increase in the radiation dose delivered to target tissues. ,, Yadav et al. evaluated the salvage therapeutic value of the ¹⁷⁷Lu-DOTAGA.(SA.FAPi)₂ dimer in 19 patients with end-stage metastatic breast cancer who had extremely limited treatment options. The study demonstrated a clinical disease control rate of 95% and an objective response rate of 84%, with a median progression-free survival of 8.5 months and a median overall survival of 12 months. Furthermore, significant pain relief was observed in over 90% of patients, underscoring the substantial palliative benefit of this approach. These findings confirm the safety and clinical efficacy of the dimer therapy by high disease burden and poor prognosis, providing pivotal evidence for the application of FAP-targeted radionuclide therapy in refractory breast cancer.

8. Current Challenges and Perspective

The development of FAPI multimers, particularly dimer and heterodimers, marks a significant advancement in nuclear medicine theranostics.Compared with traditional monomeric ligands, dimers exhibit significantly improved tumor residence time, optimized pharmacokinetics, and enhanced affinity and sensitivity. Heterodimeric strategies further amplify their utility by effectively countering tumor heterogeneity. These attributes facilitate higher sensitivity in detecting small lesions and metastases via SPECT and PET, while demonstrating unique value in delayed imaging. By leveraging the polyvalency effect, these probes successfully address the critical limitation of rapid tumor washout associated with monomeric FAPIs, thereby unlocking their therapeutic potential (Figure ). However, FAPI dimer probes currently face several challenges that need to be addressed for their clinical application. These challenges include:

• (1)Nontarget organs and dose-limiting toxicity. Due to renal excretion, high and persistent kidney uptake represents a primary dose-limiting factor and a potential risk for radionuclide therapy, necessitating the exploration of renal protection strategies.
• (2)The complexity of molecular structure optimization presents a significant hurdle. Finding the optimal balance of linker length, rigidity, and chemical properties plays a decisive role in determining overall pharmacokinetics. This optimization process is highly challenging and requires extensive systematic research. Furthermore, increased R&D costs and production difficulties are major concerns. The more complex structure of dimers imposes higher requirements on their synthesis, purification, quality control, and the pathway to clinical translation.
• (3)Increased uptake in excretory organs. The enhanced tumor retention and increased molecular weight of dimers can lead to elevated and prolonged uptake in excretory organs, notably the kidneys, posing a potential risk for nephrotoxicity during RLT. This necessitates ongoing optimization of probe design (e.g., fine-tuning linker hydrophilicity) and exploration of renal protection strategies. The synthetic complexity and higher production costs of multimers also present hurdles for widespread clinical adoption.

4.

Mechanisms of FAPI dimerization and the polyvalency effect.

In conclusion, while FAPI dimeric and heterodimeric probes have successfully demonstrated superior diagnostic sensitivity and potent therapeutic efficacy across a broad spectrum of preclinical models and in early clinical trials,several challenges related to renal dosimetry, synthetic complexity, and cost need to be addressed to realize Clinical translation of the probe. Overcome limitations such as uptake in nontarget organs, dose-limiting toxicity and elevated and prolonged uptake in excretory organs will require continued innovations in molecular design. Through ongoing innovations in chemical structure of the molecule design, strategic combination therapies, and expansion into nononcologic diseases, FAPI probes are poised to solidify their role as transformative, versatile tools in precision theranostics, ultimately improving outcomes for patients across a wide disease spectrum.

With the increasing pace of radiopharmaceutical approvals, the field of FAP-targeted therapies is undergoing rapid development. First-generation monomeric tracers such as FAPI-46 have largely transitioned into industry-led clinical development and are currently being evaluated in company-sponsored clinical trials, the focus is increasingly shifting toward next-generation probe. Specifically, distinguished by their extended tumor retention and enhanced therapeutic potential, these dimers have attracted significant scientific interest. Notably, several premier academic centers have launched early phase clinical investigations, aiming to achieve a substantial breakthrough in FAPI-based theranostics by optimizing pharmacokinetics.

Our review underscores that key design parameters-linker chemistry (length, flexibility, cleavability, hydrophilicity), chelator selection, and radionuclide pairing-are not merely structural details but are decisive for optimizing pharmacokinetics, tumor retention, and safety profiles. The clinical success of heterodimers like FAPI-RGD and PSFA-01 validates the strategy of dual-targeting to overcome tumor heterogeneity, significantly improving diagnostic sensitivity and coverage. The translation of FAPI dimers into theranostic applications is particularly promising. The ability to use chemically identical ligands for both imaging (with ⁶⁸Ga) and therapy (with ¹⁷⁷Lu) epitomizes precision medicine. Early clinical results in refractory cancers, such as metastatic breast cancer, are encouraging and justify larger, controlled trials.

Future directions are multifaceted. First, the exploration of higher-order multimers (trimers, tetramers) and novel heterodimeric combinations for specific cancer subtypes holds promise. Second, the development of multifunctional probes that integrate diagnostic, therapeutic, and real-time imaging (e.g., fluorescence) capabilities is an exciting frontier. Third, combination therapies, such as coupling FAPI-targeted RLT with immunotherapy (e.g., PD-L1 inhibitors), have shown synergistic efficacy in preclinical models and represent a highly active research area. Finally, the expansion into nononcologic fields like fibrosis and arthritis demonstrates the versatile potential of FAPI-targeted probes beyond oncology. The optimization of probes and the ultimate realization of their clinical application in theranostics rely heavily on multidisciplinary collaboration.

Glossary

Abbreviations

FAP

Fibroblast activation protein

FAPI

Fibroblast activation protein inhibitor

TME

The tumor microenvironment

CAFs

Cancer-Associated Fibroblasts

ECM

Extracellular Matrix

RLT

radioligand therapy

TRB

tumor-to-background ratios

PET

positron emission tomography

SPECT

single-photon emission computed tomography

PEGs

Polyethylene Glycols

NPC

nasopharyngeal carcinoma

TBR

tumor-to-background ratios

GC

Gastric cancer

RCC

Renal cell carcinoma

ILD

Interstitial lung disease

RA

Rheumatoid arthritis.

Peng Jiang contributed to literature search, and initial drafting. Kejing Shao contributed to data extraction. Bao Zhu and Fei Chen conceived the review, supervised the work, performed critical revision, and finalized the manuscript. All authors reviewed and approved the final version.

The authors declare no competing financial interest.