Research ArticleBasic Science Investigation

Design of a Fibroblast Activation Protein–Targeted Radiopharmaceutical Therapy with High Tumor–to–Healthy-Tissue Ratios

Ramesh Mukkamala, Daniel J. Carlson, Nicholas Kaine Miller, Spencer D. Lindeman, Emily Renee Bowen, Pooja Tudi, Taylor Schleinkofer, Owen C. Booth, Abigail Cox, Madduri Srinivasarao and Philip S. Low
Journal of Nuclear Medicine August 2024, 65 (8) 1257-1263; DOI: https://doi.org/10.2967/jnumed.124.267756

Abstract

Because of upregulated expression on cancer-associated fibroblasts, fibroblast activation protein (FAP) has emerged as an attractive biomarker for the imaging and therapy of solid tumors. Although many FAP ligands have already been developed for radiopharmaceutical therapies (RPTs), most suffer from inadequate tumor uptake, insufficient tumor residence times, or off-target accumulation in healthy tissues, suggesting a need for further improvements. Methods: A new FAP-targeted RPT with a novel ligand (FAP8-PEG3-IP-DOTA) was designed by combining the desirable features of several previous ligand-targeted RPTs. Uptake and retention of [111In]In or [177Lu]Lu-FAP8-PEG3-IP-DOTA were assessed in KB, HT29, MDA-MB-231, and 4T1 murine tumor models by radioimaging or ex vivo biodistribution analyses. Radiotherapeutic potencies and gross toxicities were also investigated by monitoring tumor growth, body weight, and tissue damage in tumor-bearing mice. Results: FAP8-PEG3-IP-DOTA exhibited high affinity (half-maximal inhibitory concentration, 1.6 nM) and good selectivity for FAP relative to its closest homologs, prolyl oligopeptidase (half-maximal inhibitory concentration, ∼14.0 nM) and dipeptidyl peptidase-IV (half-maximal inhibitory concentration, ∼860 nM). SPECT/CT scans exhibited high retention in 2 different solid tumor models and minimal uptake in healthy tissues. Quantitative biodistribution analyses revealed tumor–to–healthy-tissue ratios of more than 5 times for all major organs, and live animal studies demonstrated 65%–93% suppression of tumor growth in all 4 models tested, with minimal or no evidence of systemic toxicity. Conclusion: We conclude that [177Lu]Lu-FAP8-PEG3-IP-DOTA constitutes a promising and safe RPT candidate for FAPα-targeted radionuclide therapy of solid tumors.

Fibroblast activation protein α (FAP) is a serine protease that is overexpressed on cancer-associated fibroblasts in about 90% of human epithelial tumors (1). Because FAP is minimally expressed in healthy tissues (1), the cell surface receptor has become an attractive biomarker for the diagnosis and treatment of human cancers. Indeed, a FAP-targeted 68Ga-radioimaging agent has been recently shown to image 28 different human cancer types (2), and multiple FAP-targeted radioligands have entered clinical trials for imaging or therapy of solid tumors (3–7). However, whereas preclinical development of FAP-targeted radioligands has been steadily expanding, their radiotherapeutic potencies have frequently suffered from insufficient tumor uptake, inadequate tumor residence times, or unacceptable accumulation in the healthy tissues, especially when physiologically relevant tumor models have been used (8–10). Although we and others have attempted to address these inadequacies with improved versions of FAP-targeted radiopharmaceutical therapies (RPTs) (11–13), none have achieved the tumor–to–healthy-tissue dosimetry ratios needed to eradicate tumors without causing significant off-target damage (14,15).

We report here the design, synthesis, and evaluation in vitro and in vivo of a new FAP-targeted radioligand (FAP8-PEG3-IP-DOTA) that exhibits superior properties to previous candidates. To achieve these improvements, we have incorporated the optimal features of 3 ligand-targeted RPTs described by others, namely the basic ligand scaffold of Šimková et al. (16), which reports picomolar affinity for FAP; the gem-difluoro modification of Jansen et al. (17), which endows their compound 60 with excellent ligand specificity for FAP over prolyl oligopeptidase (PREP); and the albumin-binding moiety of the radiofolate cm09 of Müller et al., which increases its pharmacokinetics and accumulation in solid tumors (18). We then demonstrate that this novel trifunctional conjugate, FAP8-PEG3-IP-DOTA, exhibits the improved affinity, enhanced organ specificity, elevated tumor accumulation, and reduced healthy-tissue toxicity expected of the composite RPT and propose that it should qualify for possible preclinical development.

MATERIALS AND METHODS

Synthetic schemes for FAP8 (2), FAP8-PEG3-IP-DOTA (3), FAP8-PEG3-DOTA (4), and FAP8-PEG3-FITC (5) are given in Figure 1, with complete experimental procedures presented in Supplemental Schemes 1–4 (supplemental materials are available at http://jnm.snmjournals.org). Protocol details for all studies are also presented in the supplemental material (19–20).

FIGURE 1.
FIGURE 1.

Chemical structures of inhibitor (1) (16), FAP-targeting ligand (FAP8) (2), FAP8-PEG3-IP-DOTA (3), and FAP8-PEG3-DOTA (4).

Cell Culture

Cancer cell lines 4T1, KB, HT29, and MDA-MB-231 were purchased from ATCC and cultured as formerly reported (21). HEK-293T cells transduced to express high levels of human FAP (HEK-hFAP) were previously generated (22).

Radiolabeling

FAP8-PEG3-DOTA or FAP8-PEG3-IP-DOTA was dissolved in NH4OAc buffer (1.0 M, pH 7.0) and labeled with [111In]In3+ (BWMX Canada) or [177Lu]Lu3+ (National Isotope Development Center).

Animal Husbandry

Mice were provided normal rodent chow and water ad libitum and maintained on a standard 12-h light–dark cycle. All animal procedures were approved by the Purdue Animal Care and Use Committee.

Tumor Models

BALB/c mice were inoculated on their shoulders with 1 × 105 4T1 cells, whereas nu/nu mice were inoculated on their shoulders with 5 × 106 HT29, KB, MDA-MB-231, or HEK-hFAP cells.

SPECT/CT Scans

HT29 and KB tumor–bearing mice were injected intravenously with [111In]In-FAP8-PEG3-IP-DOTA (∼15 MBq/mouse), and SPECT/CT scans were taken at multiple time points over the course of 1 wk.

Ex Vivo Radioligand Biodistribution

Mice implanted with 4T1 or HEK-hFAP tumors were intravenously injected with [177Lu]Lu-FAP8-PEG3-IP-DOTA, and ex vivo biodistribution measurements of select organs were taken at several time points.

Dosimetry Analysis

From the biodistribution data, the total absorbed radiation doses (mGy/MBq) were calculated using OLINDA 2.2.3 software as previously described (11,23).

Radiopharmaceutical Therapy

4T1, HT29, KB, and MDA-MB231 tumor–bearing mice were randomized into control and treatment groups to ensure similar average starting tumor volumes. Each cohort received a single intravenous injection of saline or [177Lu]Lu-FAP8-PEG3-IP-DOTA on day 0, as indicated.

Toxicology

Mice were weighed every other day during RPT to evaluate gross toxicity. Tissue sections from organs of interest (1–8/organ per mouse) were preserved and examined for lesions in a masked manner by a board-certified veterinary pathologist.

Statistical Analysis

Data were analyzed using GraphPad Prism 9 unless otherwise stated. All results are presented as mean ± SE.

RESULTS

Evaluation of Binding Affinity and Specificity of FAP8 Conjugates

To determine the binding affinity and specificity of the new FAP8 ligand (compound 2) and FAP8-PEG3-IP-DOTA conjugate (compound 3) (Fig. 1), we measured the inhibition potency of each compound against FAP and its closest homologs, PREP and DPP-IV. As detailed in Figure 2A and Supplemental Table 1, FAP8 preferentially inhibited FAP over PREP and DPP-IV, with half-maximal inhibitory concentrations of 0.76, 13, and more than 3,000 nM, respectively, whereas FAP8-PEG3-IP-DOTA exhibited half-maximal inhibitory concentrations for the same 3 peptidases of 1.6, 14, and 860 nM, respectively (Fig. 2B). Introduction of the gem-difluoro moiety onto the proline of FAP8 ligand improved its selectivity for FAP over PREP by 7.5-fold relative to inhibitor 1 (16) as anticipated. The same modification also stabilized the linear form of inhibitor 1 over its cyclized tautomer, thereby increasing its affinity for FAP. Moreover, analysis of the direct binding of fluorescent (FAP8-PEG3-FITC) (5) (Supplemental Scheme 2) and radiolabeled ([177Lu]Lu-FAP8-PEG3-IP-DOTA) FAP8 conjugates to HEK-hFAP cells revealed dissociation constants of about 1.2 and 3.0 nM, respectively (Figs. 2C and 2D). Because this binding could be significantly inhibited on coincubation with excess unlabeled FAP8 conjugate (Figs 2C and 2D), we conclude that both of these binding interactions were FAP-mediated.

FIGURE 2.
FIGURE 2.

Measurements of enzyme inhibition of FAP, PREP, and DPP-IV by FAP8 ligand (2) (A) and FAP8-PEG3-IP-DOTA (3) (B). Binding of FAP8-PEG3-FITC (C) and [177Lu] Lu-FAP8-PEG3-IP-DOTA (D) to HEK-hFAP cells.

Ex Vivo Biodistribution Analysis

Motivated by the encouraging binding affinity and specificity of FAP8 conjugates for FAP, we next evaluated the impact of an albumin binder on both the uptake and the specificity of the FAP8-PEG3-DOTA conjugate for the tumor. For this purpose, the biodistributions of [177Lu]Lu-FAP8-PEG3-IP-DOTA and [177Lu]Lu-FAP8-PEG3-DOTA were compared, where the only difference between the 2 compounds was the 4-iodophenylbutyryllysine (IP) inserted between the PEG and the DOTA (Fig. 1, structure 4). As shown in Supplemental Figure 2, insertion of the IP increased the net tumor uptake of conjugate by about 10 times while improving both the tumor-to-kidney and the tumor-to-liver ratios by more than 2 times. These results argued in favor of including the IP insertion in all further forms of FAP8 RPTs.

Next, to determine the preferred mass dose for achieving an ideal tumor–to–healthy-tissue ratio, we chelated a fixed amount of [177Lu]LuCl3 (7.4 MBq/mouse) with 3 different concentrations of FAP8-PEG3-IP-DOTA (0.3, 1.0, and 5.0 nmol/mouse) and injected the radioligands into mice bearing syngeneic 4T1 breast cancer cells in which the only FAP+ cells were derived from naturally infiltrating cancer-associated fibroblasts. Analysis of the resulting ex vivo biodistribution data revealed that mice injected with lower doses of [177Lu]Lu-FAP8-PEG3-IP-DOTA (0.3 and 1.0 nmol/mouse) exhibited slightly lower tumor uptake at early time points (5–6 percentage injected dose [%ID]/g at 1 h after injection and 5%–9 %ID/g at 4 h after injection) than did mice injected with 5 nmol/mouse (∼9 %ID/g at 1 h after injection and 11 %ID/g at 4 h after injection) (Figs. 3A–3C; Supplemental Tables 2–4). These initial tumor uptake values gradually decreased to 3.4 ± 1 %ID/g, 5 ± 1.6 %ID/g, and 2.2 ± 0.3 %ID/g for the 0.3, 1.0, and 5 nmol/mouse doses, respectively, by 168 h after injection. In mice injected with 5 nmol of FAP8-PEG3-IP-DOTA, there was rapid clearance from both the liver (decreasing from 5.3 ± 0.90 %ID/g to ∼1 %ID/g) and the kidneys (decreasing from 4.8 ± 2.4 to 0.30 ± 0.39 %ID/g by 168 h after injection), leading to a steady increase in tumor–to–healthy-tissue ratio in these organs (Fig. 3G). In contrast, mice injected with 0.3 or 1 nmol exhibited consistently higher uptake in the liver (7.1 ± 2.0 %ID/g to 4.9 ± 0.5 %ID/g), spleen (4.1 ± 1.8 to 1.9 ± 0.45 %ID/g), and kidneys (4.9 ± 3.3 to 1.6 ± 0.7 %ID/g) at all time points tested, resulting in decreased tumor–to–healthy-tissue ratios (Figs. 3E and 3F). Although tumor uptake was slightly higher for doses of 0.3 and 1.0 nmol/mouse (3.4 ± 1 %ID/g and 5 ± 1.6 %ID/g, respectively) than for 5 nmol/mouse (2.2 ± 0.3 %ID/g) at 168 h after injection, the tumor–to–healthy-tissue ratios were lower for the 0.3- and 1.0-nmol doses than for the 5-nmol dose. From this experiment, we conclude that the best tumor–to–healthy-tissue ratios were achieved when 5 nmol of FAP8-PEG3-IP-DOTA per mouse were administered.

FIGURE 3.
FIGURE 3.

(A–D) Biodistribution analyses of [177Lu]Lu-FAP8-PEG3-IP-DOTA in 4T1 (A–C) and HEK-hFAP (D) tumor–bearing mice (4/time point). (E, F, G, and H) Calculated tumor-to-healthy tissues ratios from data in panels A, B, C, and D, respectively. B.M = bone marrow.

Because most previously published data on FAP-targeted radioligands have used tumor models in which the cancer cells were artificially transduced to express FAP (e.g., HEK-hFAP or HT1080-FAP) (8,24–26), for purposes of comparison, we elected to similarly evaluate the biodistribution of [177Lu]Lu-FAP8-PEG3-IP-DOTA in a tumor model in which the cancer cells were transduced with FAP. For this purpose, HEK-hFAP tumor–bearing mice were injected intravenously with 5 nmol of FAP8-PEG3-IP-DOTA radiolabeled with 7.4 MBq of [177Lu]Lu3+. Measurement of the biodistribution at 1, 4, and 24 h after injection revealed tumor uptake of 7.8 ± 1.3 %ID/g, 10 ± 1.7 %ID/g, and 14 ± 4.6 %ID/g, respectively. This uptake further increased to 22 ± 13 %ID/g by 72 h after injection and then gradually decreased to about 14 ± 5 %ID/g by 168 h after injection (Fig. 3D). Because the total tumor uptake at 168 h was about 7 times higher than that seen in mice implanted with FAP-negative cancer cells, we conclude that nonphysiologic induction of FAP expression in cancer cells can artificially elevate the radiation doses received in a tumor mass relative to healthy tissues (Fig. 3H; Supplemental Table 5). We therefore elected to continue characterizing our FAP8 RPT only in nontransduced tumor cell lines in which FAP naturally occurs.

Comparative Dosimetry Analyses

To determine whether 5 nmol of FAP8-PEG3-IP-DOTA per mouse might saturate available FAP receptors in an average 4T1 tumor, we computed the total absorbed dose of [177Lu]Lu-FAP8-PEG3-IP-DOTA in both solid tumors and healthy tissues (area under the curve over 168 h). As shown in Figures 4A–4C and Supplemental Tables 6–7, increasing the amount of injected FAP8-PEG3-IP-DOTA by a factor of 16.7 (i.e., from 0.3 to 1.0 to 5 nmol/mouse) raised the total absorbed dose in the tumor masses only from 904 to 994 and 1,140 mGy/MBq, respectively. Because the tumor dose increased only 25% after about a 17-times increase in FAP8-PEG3-IP-DOTA concentration, we conclude that the 5 nmol/mouse dose nearly saturates the FAP receptors in 4T1 tumors. As anticipated, the total absorbed doses were approximately 2-fold higher in HEK-hFAP tumors than in 4T1 tumors (2,270 vs. 1140 mGy/MBq), whereas dosimetry for healthy tissues was more similar irrespective of the tumor model. Thus, in healthy tissues such as the kidneys and bone marrow, which are at risk for radiation toxicity (27), absorbed doses were 220 and 48 mGy/MBq in 4T1 tumor–bearing mice, respectively, but 234 and 134 mGy/MBq, respectively in HEK-hFAP tumor–bearing mice. Because these values are both substantially lower than the absorbed doses of 1,140 mGy/MBq in 4T1 tumors and 2,270 mGy/MBq in HEK-hFAP tumors, we conclude that FAP8-PEG3-IP-DOTA delivers significantly more radiation to tumors than to healthy tissues (>5 times), regardless of the tumor model used.

FIGURE 4.
FIGURE 4.

(A–D) Comparison of absorbed radiation doses by select organs in 4T1 (A–C) and HEK-hFAP (D) tumor–bearing mice. (E, F, G, and H) Calculated tumor-to-healthy tissues ratios from data in panels A, B, C, and D, respectively. B.M = bone marrow.

Radiopharmaceutical Therapy and Toxicology

Encouraged by the favorable biodistribution and dosimetry data, we proceeded to examine the therapeutic efficacy of a single dose of [177Lu]Lu-FAP8-PEG3-IP-DOTA in mice bearing human breast (MDA-MB-231), cervical (KB), and colorectal (HT29) tumor xenografts. As mentioned for the 4T1 tumors described above, MDA-MB-231, KB, and HT29 cancer cells were selected for this study because the only FAP expression in the derived tumors is found on the infiltrating fibroblasts, that is, similar to the FAP expression pattern found in human tumors (1). As shown in Figures 5A–5C, mice treated with 37 MBq of [177Lu]Lu-FAP8-PEG3-IP-DOTA exhibited reductions in tumor growth rates of 93%, 65%, and 75% in the MDA-MB-231, KB, and HT29 tumor xenograft models, respectively. Although treated mice experienced a 5%–10% body weight loss during the first week of therapy, all mice subsequently recovered and survived without any obvious health complications. Moreover, whereas our Institutional Animal Care and Use Committee regulations required euthanasia of the mice before their overall survivals could be determined, the stalled tumor growth and prolonged survival rates in all 3 models suggested that a significant improvement in overall survival might be achievable with the single RPT dose.

FIGURE 5.
FIGURE 5.

(A–C) Antitumor efficacy of [177Lu]Lu-FAP8-PEG3-IP-DOTA (37 MBq on day 0) in MDA-MB-231 (5/group) (A), KB (3/group) (B), and HT29 (5/group) (C) tumor–bearing mice. (D and E) Representative micrographs of 4-μm hematoxylin- and eosin-stained sections of fixed heart, liver, and kidney tissues after administration of vehicle (D) or [177Lu]Lu-FAP8-PEG3-IP-DOTA (E) in HT29 tumor–bearing mice. ns = nonsignificant. *P < 0.05. **P < 0.01.

To determine whether any overt toxicities were caused by [177Lu]Lu-FAP8-PEG3-IP-DOTA RPT, tissue sections from the heart, lung, liver, and kidneys of all treated and untreated control groups were examined by a board-certified veterinary pathologist after hematoxylin and eosin staining. Although no diagnostic lesions or other morphologic abnormalities were detected in the HT29 tumor–bearing mice (Figs. 5D and 5E), 2 of 5 MDA-MD-231 tumor–bearing mice and one of the KB tumor–bearing mice had liver lesions marked by a periportal mononuclear cells, mainly lymphocytes and plasma cells and localized hepatocellular necrosis (Supplemental Figs. 3–4). It was concluded that the therapy was generally safe, though the lesions were mild to moderate in severity.

Because the number of RPT doses that a cancer patient can receive is limited by the cumulative radioactive exposure of healthy tissues in a patient, we decided to determine whether tumor growth might still be suppressed by lower doses of [177Lu]Lu-FAP8-PEG3-IP-DOTA. For this purpose, 4 different specific activities of 177Lu (0, 9.25, 18.5, and 37 MBq) were administered to 4T1 tumor–bearing mice. The syngeneic 4T1 breast cancer model was selected because its more aggressive growth rate would allow for greater differentiation in responses among treatment groups. As shown in Figure 6A, tumors in untreated mice reached their maximum allowed volumes (1,500 mm3) by day 12, whereas tumors in all 3 treatment groups stopped growing before reaching this maximum allowed volume, suggesting that their tumor progression may have been measurably inhibited. Although mice in the 9.25-MBq and 18.5-MBq cohorts did not experience any body weight loss, mice treated with 37 MBq exhibited about 5%–10% body weight loss (Fig. 6B), suggesting that 9.25- and 18.5-MBq doses may be well tolerated but that higher doses may be toxic.

FIGURE 6.
FIGURE 6.

Dose response of [177Lu]Lu-FAP8-PEG3-IP-DOTA in BALB/c mice bearing 4T1 tumors (5/group). Cohorts were treated with single dose of [177Lu]Lu-FAP8-PEG3-IP-DOTA on day 0 and then measured for tumor growth (A), body weight (B), and overall survival (C). ns = nonsignificant. **P < 0.01.

Analysis of SPECT/CT Imaging in Multiple Tumor Models

Finally, because response rates to RPTs have been shown to correlate with expression levels of the targeted receptors (28), it became important to determine whether our FAP8-PEG3-IP-DOTA might also be used as an imaging agent to stratify cancer patients by FAP expression. Therefore, HT29 and KB tumor–bearing mice were intravenously injected with a 5 nmol/mouse dose of FAP8-PEG3-IP-DOTA in which the FAP8 conjugate was labeled with the SPECT imaging agent [111In]In3+ rather than the radiotherapeutic agent [177Lu]Lu3+. Whole-body SPECT/CT scans collected at various time points (Figs. 7A and 7B) revealed that [111In]In-FAP8-PEG3-IP-DOTA accumulated rapidly in both HT29 and KB tumors, where it persisted for at least 168 h. In contrast, most of the radioactivity in the healthy tissues was cleared within 24 hours after injection. Because this uptake could be blocked by coadministration of a 100-fold excess of unlabeled FAP8-PEG3-IP-DOTA, we conclude that retention of [111In]In-FAP8-PEG3-IP-DOTA in the tumors was FAP-mediated (Supplemental Figs. 5 and 6). This prolonged retention in tumors combined with only transient residence in healthy tissues portends well for the potential safety of [177Lu]Lu-FAP8-PEG3-IP-DOTA as an RPT in humans.

FIGURE 7.
FIGURE 7.

In vivo SPECT/CT imaging of [111In]In-FAP8-PEG3-IP-DOTA conjugate in mice bearing HT29 (n = 2) (A) and KB (B) (n = 2) tumors. White arrow (a) indicates tumor, red arrow (b) indicates liver, and yellow arrow (c) indicates kidneys. p.i. = after injection.

DISCUSSION

The overarching goal of this study was to improve FAP-targeted RPT tumor–to–healthy-tissue dosimetry ratios sufficiently to comply with Food and Drug Administration guidelines for radiotherapies. On the basis of external-beam radiation (not RPT) studies, the Food and Drug Administration has recommended that total cumulative radiation doses to healthy tissues be limited to less than 20 Gy for heart, less than 7 Gy for lungs, less than 30 Gy for liver, less than 23 Gy for kidneys, and less than 2–5 Gy for bone marrow (29). Because radiosensitive tumors respond at about 40 Gy whereas radioresistant tumors may require up to 100 Gy to shrink (29–31), the tumor–to–healthy-organ dosimetry ratios necessary for a safe therapeutic index can be determined. Assuming that tumor radiation doses should exceed about 100 Gy, with maximum kidney, liver, and bone marrow cumulative exposures remaining below 23, 30, and 5 Gy, respectively, a conservative estimate suggests that tumor-to-kidney, tumor-to-liver, and tumor–to–bone marrow ratios need to exceed 4.34, 3.3, and 20 times, respectively. Compliance with these minimum ratios would ensure effective tumor shrinkage without significant normal-tissue toxicity. On the basis of the above dose escalation data, a 5 nmol/mouse dose of [177Lu]Lu-FAP8-PEG3-IP-DOTA achieved a total tumor-absorbed dose of 1,140 mGy/MBq in the 4T1 mice, which was 25.3, 14.0, 4.2, 10.3, 5.2, and about 24.0 times higher than the total absorbed doses in the heart, lungs, liver, spleen, kidney, and bone marrow, respectively (Fig. 4G). We therefore conclude that [177Lu]Lu-FAP8-PEG3-IP-DOTA should meet the safety requirements for an effective RPT in humans. Although it is difficult to compare data from different labs using different FAP-transduced tumor models (25,26,32), our previous FAP6-targeted RPT did not meet these Food and Drug Administration requirements. Thus, [177Lu]Lu-FAP8-PEG3-IP-DOTA exhibited an 8-times higher total tumor dose than did FAP6-IP-DOTA conjugate in the same 4T1 tumor–bearing mouse model at the same 5 nmol/mouse concentration, and its tumor-to-kidney ratio (>5× vs. 2×) was also superior. Not surprisingly, [177Lu]Lu-FAP8-PEG3-IP-DOTA also induced greater tumor growth inhibition than did [177Lu]Lu-FAP6-IP-DOTA (80% vs. 40%), even though about 6-times less radioactivity (9.25 vs. 55 MBq) was used than for the former therapy (11).

We have also repeatedly noted that better tumor–to–healthy-tissue ratios are achieved at higher mass doses, regardless of the amount of radionuclide chelated by the RPT. We propose that this can be explained by the relative rates of perfusion of the tumor versus healthy tissues such as the lungs, kidneys, gastrointestinal tract, and liver. Although 100%, 22%, 21%, and 6% of the blood perfuses the lungs, kidneys, gastrointestinal tract, and liver, respectively (33), during each circulatory cycle through the body, only a very minor fraction (<2%) will perfuse a small solid tumor (34–36). Thus, when the administered amount of FAP8-PEG3-IP-DOTA is low (e.g., at 0.3 and 1.0 nmol/mouse), most of the individual radioligands will be captured either by scavenger receptors or by the very low numbers of FAP+ fibroblasts that may exist in healthy tissues, leaving very little [177Lu]Lu-FAP8-PEG3-IP-DOTA to perfuse the solid tumor. At higher mass doses, however, both healthy and malignant tissues can conceivably be saturated, enabling the radioligand biodistribution to reflect the relative expression levels of the targeted receptor rather than their relative rates of blood perfusion. Because FAP+ cancer-associated fibroblasts are much more abundant in solid tumors than in healthy tissues, it will be important to treat a patient with sufficient compound to nearly saturate all FAP receptors in the tumor mass and then adjust the amount of radionuclide delivered to ensure that the allowed radiation exposure of healthy tissues is not exceeded. Estimation of this amount of RPT precursor will require a dose escalation study in an appropriate tumor-bearing patient population.

We observed a significant improvement in both total tumor dose and tumor–to–healthy-tissue ratios on insertion of the albumin binder, IP, into our FAP8-PEG3-DOTA conjugate, presumably because it extended the drug’s circulatory half-life in the vasculature (37). Other groups have similarly inserted albumin binders into their FAP-targeted RPTs with the same goal of prolonging pharmacokinetics (12,13). Although the anticipated enhanced tumor uptake was achieved in all cases, these labs also reported significantly compromised tumor–to–healthy-tissue ratios, perhaps because their Evans blue albumin binder exhibited nonspecific affinity for many proteins (38). Nevertheless, one of these RPTs (177Lu-LNC1004) has shown encouraging results in a phase 1 human clinical trial (39), suggesting that perhaps the toxicities associated with low radiation exposures to healthy tissues in preclinical models may be overestimated. A limitation to our present study is the absence of a head-to-head comparison between our RPT with the other FAP-targeted RPTs currently in the clinic (e.g., FAP-2286 and FAPI-46).

CONCLUSION

We have designed a superior FAP-targeted RPT that provides tumor–to–healthy-tissue ratios that exceed the requirements established by the Food and Drug Administration, suggesting that it should be considered a viable candidate for human clinical evaluation.

DISCLOSURE

Ramesh Mukkamala, Spencer D. Lindeman, Madduri Srinivasarao, and Philip S. Low hold a patent on FAP-targeted RPT. Financial support was provided through a Purdue professorship. No other potential conflict of interest relevant to this article was reported.

KEY POINTS

QUESTION: Can a FAP-targeted radioligand demonstrate sufficient tumor–to–healthy-tissue ratios and efficacy for preclinical development in murine tumor models in which FAP expression is limited to cancer-associated fibroblasts?

PERTINENT FINDINGS: [177Lu]Lu-FAP8-PEG3-IP-DOTA exhibited superior tumor–to–healthy-tissue ratios and successfully treated multiple murine tumor models generated from FAP-negative cancer cells.

IMPLICATIONS FOR PATIENT CARE: These data suggest that [177Lu]Lu-FAP8-PEG3-IP-DOTA constitutes a promising candidate for development of FAP-targeted RPT for solid tumors.

ACKNOWLEDGMENTS

We acknowledge the Purdue Imaging Facility for its general support of the SPECT/CT studies and the assistance of MacKenzie McIntosh and the Purdue University Histology Research Laboratory. Ramesh Mukkamala thanks Charity Campbell, Mar, Brandon Chuck-Wang Mar, Jackson N. Moss, Autumn Horner, and Losha Dasol Jung for their help in select studies.

Footnotes

  • Published online Jun. 13, 2024.

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  • Received for publication March 9, 2024.
  • Accepted for publication May 13, 2024.
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