Therapeutic Efficacy of 177Lu-Labeled A20FMDV2 Peptides Targeting ανβ6

Integrin ανβ6 promotes migration and invasion of cancer cells, and its overexpression often correlates with poor survival. Therefore, targeting ανβ6 with radioactive peptides would be beneficial for cancer imaging and therapy. Previous studies have successfully developed radiotracers based on the peptide A20FMDV2 that showed good binding specificity for ανβ6. However, one concern of these ανβ6 integrin-targeting probes is that their rapid blood clearance and low tumor uptake would preclude them from being used for therapeutic purposes. In this study, albumin binders were used to increase tumor uptake for therapeutic applications while the non-albumin peptide was evaluated as a potential positron emission tomography (PET) imaging agent. All peptides used the DOTA chelator for radiolabeling with either 68Ga for imaging or 177Lu for therapy. PET imaging with [68Ga]Ga-DOTA-(PEG28)2-A20FMDV2 revealed specific tumor uptake in ανβ6-positive tumors. Albumin-binding peptides EB-DOTA-(PEG28)2-A20FMDV2 and IBA-DOTA-(PEG28)2-A20FMDV2 were radiolabeled with 177Lu. Biodistribution studies in normal mice showed longer blood circulation times for the albumin binding peptides compared to the non-albumin peptide. Therapy studies in mice demonstrated that both 177Lu-labeled albumin binding peptides resulted in significant tumor growth inhibition. We believe these are the first studies to demonstrate the therapeutic efficacy of a radiolabeled peptide targeting an ανβ6-positive tumor.


Introduction
Integrins are an important class of cell surface receptors that are responsible for cell-matrix adhesion and signaling across the membrane, therefore controlling a variety of vital cell functions such as cellular growth, proliferation, migration, signaling, and cytokine activation that are critical to infection, inflammation, and cancer [1,2]. Their diverse functions make them attractive therapeutic targets and certain integrin-targeted drugs have been effectively utilized in the clinic or in clinical trials for cancer therapy [3][4][5][6]. In recent years, integrin α ν β 6 has gained much attention due to its overexpression in various kinds of aggressive cancers and its correlation with worse prognosis and survival outcomes [7][8][9][10][11].
Several radiolabeled α ν β 6 -targeting ligands have been identified and used in preclinical imaging studies [12][13][14][15][16][17]. Quigley et al. introduced a cyclic nonapeptide c[YRGDLAYp(NMe)K] radiolabeled with 68 Ga that showed a high affinity and target-specific uptake in integrin α ν β 6 -positive tumors [16]. Kimura et al. presented a series of highly stable cystine knot peptides radiolabeled with 64 Cu that showed potent and specific integrin α v β 6 binding in vitro and in vivo studies [17]. In this study, we focus on a 20-amino-acid peptide sequence of NAVPNLRGDLQVLAQKVART (A20FMDV2) reported by Hausner et al. that showed a high target affinity and selectivity for the integrin receptor [12]. Recently, this group translated a similar peptide in which the lysine at position 16 was replaced with an arginine (A20FMDV2-K16R) for clinical imaging when radiolabeled with 18 F [18].

Therapy Studies
The anti-tumor efficacy was investigated in BxPC-3 tumor-bearing mice with a sin- Figure S4). Tumor volumes were found to be significantly reduced in comparison to the control group (p < 0.001) for both treated groups ( Figure S4). However, treated mice experienced significant weight loss of more than 20%, resulting in the death of all mice from    ) when compared to the control group (51.39 ± 5.56%) (p = 0.0044). Injury was not observed in the heart, lung, liver and spleen sections (data not shown).

Discussion
There is a wide prevalence of α v β 6 -integrin expression in different kinds of cancer. It is known that an elevated level of integrin α v β 6 is associated with poor prognosis as it promotes cell invasion and migration-the two crucial processes responsible for metastasis. In recent years, a number of radiolabeled α v β 6 integrin ligands for in vivo imaging and therapy of the integrin have been developed. Among these, the 20-mer peptide, A20FMDV2, has been extensively studied and radiolabeled with a variety of radionuclides ( 18 F, 68 Ga, 64 Cu, and 111 In) [26][27][28][29]. However, it has not been evaluated as of yet with therapeutic radionuclides, likely because of its short blood half-life that leads to low tumor uptake.
All radiotracers were radiolabeled in good radiochemical purity at a molar activity of 10-18.5 MBq/nmol. A high temperature of 90 • C was required for the labeling, which is consistent with previous studies, which used up to 99 • C for 68 Ga and 80-95 • C for 177 Lu labelings [35][36][37]. In vitro cell-based studies in BxPC-3 cell lines indicated that about 11% internalized for the [ 68 Ga]Ga-DOTA-(PEG28) 2 -A20FMDV2 at 1 h, while the albumin binding constructs had about 8-10%. These results are similar to what was observed for the 64 Cu-labeled constructs evaluated by Ganguly et al., in which 14.5% and 11.9% internalization was observed for their non-albumin and albumin binding peptides, respectively, in BXPC-3 cells [20]. Of course, a direct comparison is difficult due to different radionuclides, linkers, and the K16R substitution. We demonstrated good imaging of BXPC-3 tumors with [ 68 Ga]Ga-DOTA-(PEG28) 2 -A20FMDV2 that was specific as evidenced by the reduction in SUV upon administration of the blocking agent. Other 68 Ga studies targeting integrin α v β 6 have shown good tumor uptake compared to blocking and clearance through the kidney, which is consistent with our results [16,35,36,38].
Therapy studies showed that both Reducing the amount of radioactivity administered alleviated some toxicity that was seen with the 37 MBq dose while still producing a therapeutic response; however, tumor to normal tissue ratios must be improved in order to increase the therapeutic index. Therefore, further modifications to the peptide must be made to reduce the normal tissue uptake, especially for the kidney.

General Methods
All solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) and used as received. All solutions and buffers were prepared using HPLC-grade water. Radio-TLCs employed Whatman 60 Å silica gel thin-layer chromatography (TLC) plates and were analyzed using a Bioscan 200 imaging scanner (Bioscan, Inc., Washington, DC, USA). Radioactivity was counted with a Beckman Gamma 8000 counter containing a NaI crystal (Beckman Instruments, Inc., Irvine, CA, USA). The peptides EB-DOTA-(PEG28) 2 -A20FMDV2, IBA-DOTA-(PEG28) 2 -A20FMDV2, and non-albumin peptide DOTA-(PEG28) 2 -A20FMDV2 were synthesized by AnaSpec company (Fremont, CA, USA) and characterized by HPLC and mass spectrometry. A stock solution of 1 nmol/µL was made with HPLC-grade water and stored at −20 • C before use. Non-PEGylated A20FMDV2 peptide served as a blocking agent. BxPC-3 cells were purchased from ATCC (Manassas, VA, USA) and grown in RPMI 1640, 10% FBS, and 10 mM HEPES. The cells were cultured in an incubator at 37 • C, 5% CO2, and harvested in PBS by trypsin-EDTA 0.25% before use.

68 Ga Radiochemistry
68 Ga was obtained from 68 Ge/ 68 Ga generator (Eckert and Ziegler) at Mallinckrodt Institute of Radiology, Washington University School of Medicine [39]. Briefly, 68 Ga was eluted from the generator in 5mL of 0.1M HCl and collected into a vial. The resulting solution was loaded onto a strong cation Strata XC column 30 mg/mL 33 µm (Phenomenex) and the activity was retained in the column. The column was eluted with 1 mL 98% acetone (0.02M HCl) and the resulting activity was collected in a 1.5 mL Eppendorf tube. The solution was heated to evaporation at 90 • C for 15 min until 10-20 µL 68 Ga was achieved. Aqueous ammonium acetate (0.1 M, pH 5.5, 100 µL) and a solution of DOTA-(PEG28) 2 -A20FMDV2 (2 nmol) were added to a solution of 1 mCi 68 Ga. The reaction mixture was incubated at 90 • C for 15 min and evaluated for radiochemical purity by thin-Pharmaceuticals 2022, 15, 229 9 of 13 layer chromatography with the mobile phase of 50 mM DTPA. The radiolabeled complex remained at the origin while the free 68 Ga moved with the solvent front.

177 Lu Radiochemistry
177 Lu (no-carrier added) was obtained from the University of Missouri Research Reactor (MURR). For 177 Lu labelings, 177 LuCl 3 in 0.05 M HCl was mixed with NH 4 OAc 0.1M pH 5.5 in a 10:1 ratio to obtain a solution of pH 5.0-5.5. After the addition of the albumin conjugates (4-5 µL stock solution) to 0.5 mCi of 177 Lu, the reaction vial was incubated for 15 min at 90 • C. For therapy studies, 100 µL stock solution of albumin peptides were added to 10 mCi of 177 Lu. Quality control was performed by TLC as described above. The radiolabeled complexes remain at the origin while free 177 Lu moves with the solvent front. Radiolabeled products (>95% purity) were used directly without further purification.

Serum Stability
The stability of the radioligands was determined over time using radioTLCs with 50 mM DTPA as mobile phase. 10 µL of radiotracers was added to the Eppendorf tubes, each containing 100 µL of human serum or 1X PBS. The tubes were incubated at 37 • C with moderate agitation. The integrity of the compounds was investigated after incubation at 1, 3, 5, and 7 days. The experiment was conducted in triplicates and 0.5 µL aliquots of radiotracers were to be withdrawn to evaluate the amount of intact compound by TLC.

Cellular Uptake and Internalization
The total cell binding and internalized fractions were conducted using α ν β 6  Tissues of interest (blood, lung, liver, spleen, kidney, muscle, heart, bone, and tumor) were collected and weighed, and the radioactivity was measured using a gamma counter. The results were expressed as the percentage of injected dose per gram of tissue (%ID/g).
The comparison of non-albumin binding and albumin binding peptides were evaluated in CD-1 mice at 1, 4, 24, and 48 h. 177 Lu-labeled peptides were prepared at the specific activity of 10 MBq/nmol. Mice (n = 4-5) were intravenously injected with 0.37 MBq (10 µCi) of respective radioligands diluted in 100 µL saline and sacrificed at specified time points. Selected organs were collected, weighed, and counted for activity. The results were reported as the percentage of the injected radioactivity per gram of tissue mass (% ID/g) and the radioactivity was calibrated using a known standard.

PET Imaging Studies
Mice (n = 3) were injected with 3.7 MBq (100 µCi) of [ 68 Ga]Ga-DOTA-(PEG28) 2 -A20FMDV2 and PET imaging was performed 1 h after radiotracer injection. For blocking study, excess of unlabeled peptides was administered 1-2 min before radiotracer injection. The PET scans were acquired for 20 min on an Inveon small animal PET/CT scanner (Siemens Medical Solutions, Malvern, PA, USA). Static images were reconstructed with the maximum a posteriori (MAP) reconstruction algorithm and corrected for decay. Image analysis was performed using the Inveon Research Workstation image display software (Siemens). Regions of interest (ROI) were selected based on co-registered anatomical CT images, and the average or maximum standard uptake value (SUV) was calculated as the mean or maximum regional radioactivity concentration (nCi/cc) x animal weight (g)/decay-corrected amount of injected dose (nCi).

Therapy Studies
Therapy studies were conducted in athymic nude mice bearing BxPC-3 tumors when the tumor volume reached about 100 mm 3 . An initial therapy study was conducted with control mice (n = 8) injected with saline and treated mice received either 37 MBq of [ 177 Lu]Lu-EB-DOTA-(PEG28) 2 -A20FMDV2 or [ 177 Lu]Lu-IBA-DOTA-(PEG28) 2 -A20FMDV2. Tumor volumes and body weights were monitored three times a week. Individual tumor size was calculated using the formula (length × width × width)/2. The mice were euthanized when the tumor reached 1500 mm 3 , ulceration of >4 mm was present, or significant stress due to weight loss. A second therapy study was conducted in BxPC-3 tumor-bearing mice in which mice (n = 8) were given either saline, 18

Immunohistochemical Staining
Tumor, kidney, heart, lung, liver, and spleen of mice (n = 4) given either saline or 37 MBq dose of [ 177 Lu]Lu-IBA-DOTA-(PEG28) 2 -A20FMDV2 were collected and fixed in neutral buffered formalin. After fixation and dehydration, tissue samples were embedded in paraffin, and 5 µm tissue sections were then stained with hematoxylin and eosin (H&E). H&E staining of tissue samples was prepared by the Anatomic and Molecular Pathology Core Lab, Washington University in St Louis. Tumors were also stained with Ki-67 for further microscopic examination. Before staining, slides were baked in 55 • C oven for 60 min to deparaffinize and heat-induced antigen retrieval was performed in citrate buffer 0.1 M pH 6.0 at 92 • C for 20 min to recover the antigens that may have been altered by fixation. Tissues were first blocked with Dako Endogenous Enzyme Block (Dako North America Inc., Carpinteria, CA, USA) for 10 min, followed by another blocking step with 10% goat serum in PBS for another 45 min. A primary antibody Ki-67 (9027, Cell Signaling, 1:100 dilution) was applied to the slides overnight at 4 • C. The secondary antibody ImmPRESS Goat Anti-rabbit (Vector Laboratories Inc., Mountain View, CA, USA) was then added for 45 min. The color was developed using DAB substrate Chromogen (Dako) and the sections were counterstained with Hematoxylin to visualize nuclei and overall tissue architecture. Sections were dehydrated, mounted, and cover-slipped. Staining results were assessed by Olympus microscope (BX51) and the cell Sense software.

Statistical Analysis
Quantitative data were processed by Prism 9 (GraphPad Software, La Jolla, CA, USA) and expressed as Mean ± SD. For therapy studies, tumor measurements and body weight changes were expressed as Mean ± SEM. Statistical analysis was performed using Student's t-test. Differences at the 95% confidence level (p < 0.05) were considered statistically significant.

Conclusions
Here, we demonstrate the first targeted radiopharmaceutical therapy for tumors expressing α v β 6 integrin. . This led to significantly greater tumor inhibition, which was not present when using [ 177 Lu]Lu-DOTA-(PEG28) 2 -A20FMDV2 at the same doses. Toxicity due to increased uptake in normal tissues remains a concern as it may lead to a narrower therapeutic index. It is anticipated that the K16R substitution that has been recently described would have a significant impact on reducing normal tissue uptake and therefore toxicity. Future studies will evaluate this substitution with the IBA albumin binder and 177 Lu to determine if the therapeutic index has been improved.  Table S1: Tumor-tonormal-organ ratios of [ 177 Lu]Lu-EB-DOTA-(PEG28) 2 -A20FMDV2 in α v β 6 -positive BxPC-3 tumorbearing mice; Table S2: Tumor-to-normal-organ ratios of [ 177 Lu]Lu-IBA-DOTA-(PEG28) 2 -A20FMDV2 in α v β 6 -positive BxPC-3 tumor-bearing mice.