*3.3. Integrin αvβ<sup>6</sup> Affinity ELISA*

Competitive ELISA against biotinylated LAP, demonstrated that both ABM modifications of αvβ6-BP were well tolerated; [NatCu]**1** and [NatCu]**2** showed high integrin αvβ6-affinity as expressed by the half-maximum inhibitory concentrations (IC50); [NatCu]**1** and [NatCu]**2**: IC<sup>50</sup> = 14 <sup>±</sup> 2 and 19 <sup>±</sup> 5 nM, respectively) compared to DOTA-αvβ6-BP (IC<sup>50</sup> = 28 ± 3 nM) [44].

#### *3.4. Cell Binding and Internalization Assay*

Cell binding studies showed that [64Cu]**1** and [64Cu]**2** both bound to cells in an <sup>α</sup>vβ6-dependent manner at similar levels (DX3puroβ6 (+): [64Cu]**<sup>1</sup>** 55.8 <sup>±</sup> 3.0% of total radioactivity, [64Cu]**<sup>2</sup>** 60.2 <sup>±</sup> 3.9%; BxPC-3 (+): [64Cu]1 30.3 <sup>±</sup> 2.7%, [64Cu]2 48.5 <sup>±</sup> 3.5%; and the negative control DX3puro (−): [64Cu]**<sup>1</sup>** 2.7 <sup>±</sup> 0.5%, [64Cu]**<sup>2</sup>** 3.1 <sup>±</sup> 0.3%, Figure 2). This resulted in binding ratios for DX3puroβ6 (+) vs. DX3puro (−) of 20.7:1 for [64Cu]**<sup>1</sup>** and 19.4:1 for [64Cu]**2**. Internalization into αvβ6-positive cells was also high ([64Cu]**1**: 48.5–52.7% of the bound radioactivity, [64Cu]**2**: 41.5–54.8%, Figure 2). The non-targeting control ABM conjugates [64Cu]**3** and [64Cu]**4** exhibited low, non-specific binding to all cell lines (≤4.3%; Figure S24).

**Figure 2.** Cell binding () and internalization () for [64Cu]Cu DOTA-EB-αvβ<sup>6</sup> -BP ([64Cu]**1**) and [ <sup>64</sup>Cu]Cu DOTA-IP-αvβ<sup>6</sup> -BP ([64Cu]**2**) for (**A**) DX3puroβ6 (αvβ6+) and DX3puro(αvβ6−) cells and (**B**) BxPC-3 (αvβ6+) cells.

#### *3.5. Human and Mouse Serum Binding Assay and Stability Assay*

Serum albumin binding for [64Cu]**1** and [64Cu]**2** was similar, with higher binding to human serum protein (53.4 ± 0.9% and 63.3 ± 1.5%, respectively) than to mouse serum protein (41.9 ± 1.1% and 44.0 ± 0.1%, respectively; Figure 3A). The ABM modifications of [ <sup>64</sup>Cu]**1** and [64Cu]**2** increased the serum albumin affinity as the [64Cu]Cu DOTA-αvβ6-BP without an ABM modification showed <29% binding to either serum albumin [44]. Both peptides showed high stability in human serum at 37 ◦C ([64Cu]**1** 1 h: 99% and 4 h: 89% intact; [64Cu]**2** 1 h: 99% and 4 h: 93% intact) with some degradation apparent after 24 h ([64Cu]**1**: 76% intact vs. [64Cu]**2**: 90% intact, Figure 3B). In contrast, faster degradation was observed in mouse serum at 37 ◦C, and the stability was lower for [64Cu]**1** than for [ <sup>64</sup>Cu]**2** at all-time points; [64Cu]**1** was 78% intact at 1 h, dropping to 58% at 4 h, and largely metabolized at 24 h (14% intact). By comparison, [64Cu]**2** was 92% and 83% intact at 1 h and 4 h, respectively, with 48% remaining intact at 24 h, a 3.4-fold higher stability than [ <sup>64</sup>Cu]**1** (Figure 3C).

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*3.6. Biodistribution*

ent non-ABM containing [

[

[

[

mulation by >3-to-4.5-fold compared to the [

ity in human serum at 37 °C. (**C**) Stability in mouse serum at 37 °C for [<sup>64</sup>Cu]**1** (■) and [

double that of [64Cu]**1** at the earliest time point, but both peptides dropped over time to below 3.2% ID/g at 72 h. The liver uptake was moderate (<3% ID/g) throughout for both peptides; but it increased to significantly higher levels for the EB-ABM containing peptide

<sup>64</sup>Cu]**1,** beginning at 24 h, reaching >1.8-fold higher levels than [64Cu]**2** at 72 h (2.36 ± 0.51% ID/g vs. 1.30 ± 0.13% ID/g, respectively; *p* = 0.025, Figure 4). Overall, the EB-ABM containing peptide [64Cu]**1** had a less favorable pharmacokinetic profile with significantly higher uptake in the kidneys and liver, resulting in generally lower tumor-to-tissue ratios for

<sup>64</sup>Cu]**1** compared to [64Cu]**2**, most notably for the tumor-to-kidney ratio ([64Cu]**1** 0.13 ± 0.06/1 to 0.19 ± 0.08/1 vs. [64Cu]**2** 0.20 ± 0.06/1 to 0.44 ± 0.14/1), and the tumor-to-liver ratio ([64Cu]**1** 2.39 ± 0.59/1 to 1.47 ± 0.47/1 vs. [64Cu]**2** 2.72 ± 0.62/1 to 3.77 ± 0.72/1) (Figure S22).

**Figure 3.** (**A**) Binding to human and mouse serum (*n* = 3/compound/condition; bars: SD). (**B**) Stability in human serum at 37 ◦C. (**C**) Stability in mouse serum at 37 ◦C for [64Cu]**1** () and [ <sup>64</sup>Cu]**2** ( **Figure 3.** (**A**) Binding to human and mouse serum (*n* = 3/compound/condition; bars: SD). (**B**) Stabil-<sup>64</sup>Cu]**2** (■). ).

#### *3.6. Biodistribution*

The biodistributions for [64Cu]**1** and [64Cu]**2** in the BxPC-3 tumor model showed good tumor uptake (4 h to 72 h: [64Cu]**1** 5.29 ± 0.59 to 3.32 ± 0.46% ID/g, [64Cu]**2** 7.60 ± 0.43 to 4.91 ± 1.19% ID/g, Figure 4). Overall, tumor uptake of [64Cu]**2** appeared higher than of [64Cu]**1**, particularly at the earliest time point, and relative tumor washout over the total observed time frame was similar for both peptides. The ABM modifications increased tumor accu-<sup>64</sup>Cu]Cu DOTA-αvβ6-BP without an ABM, which had only 1.61 ± 0.70% ID/g at 4 h in the same BxPC-3 tumor model [44]. Clearance for [64Cu]**1** and [64Cu]**2** was primarily renal and the kidneys were the organ with the highest levels of radioactivity (Figures S20-S21). Notably, [64Cu]**1** showed more than double the kidney uptake of [64Cu]**2** at 4 h, p.i. ([64Cu]**1** 75.51 ± 7.26% ID/g; [64Cu]**2** 33.56 ± 5.39% ID/g; *p* = 0.0013) and remained significantly higher for at least 48 h (>1.7-fold, *p* < 0.05), but both were cleared from the kidneys over time with accumulation dropping at 72 h ([64Cu]**1** 19.97 ± 6.91% ID/g; [64Cu]**2** 11.48 ± 1.02% ID/g; *p* = 0.103, Figure 4). Kidney accumulation for the ABM containing peptides [64Cu]**1** and [64Cu]**2** was initially higher than for the par-<sup>64</sup>Cu]Cu DOTA-αvβ6-BP (20.37 ± 1.67% ID/g at 4 h to 6.81 ± 1.36% ID/g at 48 h) [44]. Some clearance for [64Cu]**1** and [64Cu]**2** was also observed through the gastrointestinal tract (GI), with the stomach having the highest uptake at 4 h, p.i. ([64Cu]**1**: stomach 6.41 ± 0.64% ID/g, small intestines 4.72 ± 0.55% ID/g, large intestines 4.13 ± 0.10% ID/g, [64Cu]**2**: stomach 18.07 ± 2.91% ID/g, small intestines 9.55 ± 1.21% ID/g, large intestines 9.83 ± 0.69% ID/g; Figure 4 ). Clearance from the GI tract was further confirmed by radioactivity measurements of fecal matter (4–72 h: [64Cu]**1**: 3.03 ± 0.67 to 1.81 ± 0.74% ID/g; <sup>64</sup>Cu]**2**: 9.32 ± 1.08 to 2.29 ± 0.53% ID/g; Figure 4). The GI uptake for [64Cu]**2** was more than The biodistributions for [64Cu]**1** and [64Cu]**2** in the BxPC-3 tumor model showed good tumor uptake (4 h to 72 h: [64Cu]**<sup>1</sup>** 5.29 <sup>±</sup> 0.59 to 3.32 <sup>±</sup> 0.46% ID/g, [64Cu]**<sup>2</sup>** 7.60 <sup>±</sup> 0.43 to 4.91 <sup>±</sup> 1.19% ID/g, Figure 4). Overall, tumor uptake of [64Cu]**<sup>2</sup>** appeared higher than of [ <sup>64</sup>Cu]**1**, particularly at the earliest time point, and relative tumor washout over the total observed time frame was similar for both peptides. The ABM modifications increased tumor accumulation by >3-to-4.5-fold compared to the [64Cu]Cu DOTA-αvβ6-BP without an ABM, which had only 1.61 ± 0.70% ID/g at 4 h in the same BxPC-3 tumor model [44]. Clearance for [64Cu]**1** and [64Cu]**2** was primarily renal and the kidneys were the organ with the highest levels of radioactivity (Figure S20 and S21). Notably, [64Cu]**1** showed more than double the kidney uptake of [64Cu]**<sup>2</sup>** at 4 h, p.i. ([64Cu]**<sup>1</sup>** 75.51 <sup>±</sup> 7.26% ID/g; [64Cu]**<sup>2</sup>** 33.56 ± 5.39% ID/g; *p* = 0.0013) and remained significantly higher for at least 48 h (>1.7-fold, *p* < 0.05), but both were cleared from the kidneys over time with accumulation dropping at 72 h ([64Cu]**<sup>1</sup>** 19.97 <sup>±</sup> 6.91% ID/g; [64Cu]**<sup>2</sup>** 11.48 <sup>±</sup> 1.02% ID/g; *<sup>p</sup>* = 0.103, Figure 4). Kidney accumulation for the ABM containing peptides [64Cu]**1** and [64Cu]**2** was initially higher than for the parent non-ABM containing [64Cu]Cu DOTA-αvβ6-BP (20.37 <sup>±</sup> 1.67% ID/g at 4 h to 6.81 <sup>±</sup> 1.36% ID/g at 48 h) [44]. Some clearance for [64Cu]**<sup>1</sup>** and [64Cu]**<sup>2</sup>** was also observed through the gastrointestinal tract (GI), with the stomach having the highest uptake at 4 h, p.i. ([64Cu]**1**: stomach 6.41 <sup>±</sup> 0.64% ID/g, small intestines 4.72 <sup>±</sup> 0.55% ID/g, large intestines 4.13 <sup>±</sup> 0.10% ID/g, [64Cu]**2**: stomach 18.07 <sup>±</sup> 2.91% ID/g, small intestines 9.55 ± 1.21% ID/g, large intestines 9.83 ± 0.69% ID/g; Figure 4). Clearance from the GI tract was further confirmed by radioactivity measurements of fecal matter (4–72 h: [64Cu]**1**: 3.03 <sup>±</sup> 0.67 to 1.81 <sup>±</sup> 0.74% ID/g; [64Cu]**2**: 9.32 <sup>±</sup> 1.08 to 2.29 <sup>±</sup> 0.53% ID/g; Figure 4). The GI uptake for [64Cu]**2** was more than double that of [64Cu]**1** at the earliest time point, but both peptides dropped over time to below 3.2% ID/g at 72 h. The liver uptake was moderate (<3% ID/g) throughout for both peptides; but it increased to significantly higher levels for the EB-ABM containing peptide [64Cu]**1,** beginning at 24 h, reaching >1.8-fold higher levels than [64Cu]**<sup>2</sup>** at 72 h (2.36 <sup>±</sup> 0.51% ID/g vs. 1.30 <sup>±</sup> 0.13% ID/g, respectively; *p* = 0.025, Figure 4). Overall, the EB-ABM containing peptide [64Cu]**1** had a less favorable pharmacokinetic profile with significantly higher uptake in the kidneys and liver, resulting in generally lower tumor-to-tissue ratios for [64Cu]**1** compared to [64Cu]**2**, most notably for the tumor-to-kidney ratio ([64Cu]**<sup>1</sup>** 0.13 <sup>±</sup> 0.06/1 to 0.19 <sup>±</sup> 0.08/1 vs. [64Cu]**<sup>2</sup>** 0.20 <sup>±</sup> 0.06/1 to 0.44 <sup>±</sup> 0.14/1), and the tumor-to-liver ratio ([64Cu]**<sup>1</sup>** 2.39 <sup>±</sup> 0.59/1 to 1.47 <sup>±</sup> 0.47/1 vs. [ <sup>64</sup>Cu]**<sup>2</sup>** 2.72 <sup>±</sup> 0.62/1 to 3.77 <sup>±</sup> 0.72/1) (Figure S22).

*3.6. Biodistribution*

ent non-ABM containing [

[

[

[

**Figure 4.** Biodistribution time activity plots for [64Cu]**1** () and [64Cu]**2** ( ity in human serum at 37 °C. (**C**) Stability in mouse serum at 37 °C for [<sup>64</sup>Cu]**1** (■) and [ <sup>64</sup>Cu]**2** (■). ). (**A**) BxPC-3 tumors. (**B**) kidneys. (**C**) liver. (**D**) stomach. (**E**) small intestines. (**F**) large intestines (\* *p* ≤ 0.05).

The biodistributions for [64Cu]**1** and [64Cu]**2** in the BxPC-3 tumor model showed good tumor uptake (4 h to 72 h: [64Cu]**1** 5.29 ± 0.59 to 3.32 ± 0.46% ID/g, [64Cu]**2** 7.60 ± 0.43 to 4.91 ± 1.19% ID/g, Figure 4). Overall, tumor uptake of [64Cu]**2** appeared higher than of [64Cu]**1**, particularly at the earliest time point, and relative tumor washout over the total observed time frame was similar for both peptides. The ABM modifications increased tumor accumulation by >3-to-4.5-fold compared to the [ <sup>64</sup>Cu]Cu DOTA-αvβ6-BP without an ABM, which had only 1.61 ± 0.70% ID/g at 4 h in the same BxPC-3 tumor model [44]. Clearance for [64Cu]**1** and [64Cu]**2** was primarily renal and the kidneys were the organ with the highest levels of radioactivity (Figures S20-S21). Notably, [64Cu]**1** showed more than double the kidney uptake of [64Cu]**2** at 4 h, p.i. ([64Cu]**1** 75.51 ± 7.26% ID/g; [64Cu]**2** 33.56 ± 5.39% The non-αvβ6-targeting ABM controls [64Cu]**3** and [64Cu]**4** were used to determine non-specific uptake and provide support that the enhanced tumor accumulation of ABM containing peptides [64Cu]**1** and [64Cu]**2** was due to integrin αvβ<sup>6</sup> receptor mediated uptake. The biodistributions of the non-αvβ6-targeting ABM controls [64Cu]**3** and [64Cu]**4** at 4 h p.i. (*n* = 2/compound) showed prolonged blood circulation with much higher blood accumulation (38.9 ± 10.4% ID/g and 9.5 ± 1.3% ID/g, respectively; Figure 5, Figure S25). This increased blood accumulation also led to higher systemic accumulation in other tissues, especially the highly perfused tissues such as the heart, muscle, liver, and lung (Figure 5, Figure S25), with the exception of the kidneys (18.6 ± 1.4% ID/g and 4.34 ± 0.61% ID/g, respectively). These distinctly different pharmacokinetic profiles of the non-integrin αvβ6 targeting [64Cu]**3** and [64Cu]**4** resulted in a low tumor-to-blood ratio of <0.9/1 compared to >4/1 for [64Cu]**1** and [64Cu]**2**, a lower tumor-to-muscle ratio ranging from 5.6 to 6.3/1 for [ <sup>64</sup>Cu]**3** and [64Cu]**4** compared to >8/1 for [64Cu]**1** and [64Cu]**2**, and a lower tumor-to-liver ratio of 1.2–1.3/1 for [64Cu]**3** and [64Cu]**4** compared to 3.2–4.9/1 for [64Cu]**1** and [64Cu]**2** (Figure 5, Figure S26).

#### ID/g; *p* = 0.0013) and remained significantly higher for at least 48 h (>1.7-fold, *p* < 0.05), but *3.7. Blocking Biodistribution*

ID/g at 48 h) [44]. Some clearance for [64Cu]**1** and [64Cu]**2** was also observed through the gastrointestinal tract (GI), with the stomach having the highest uptake at 4 h, p.i. ([64Cu]**1**: stomach 6.41 ± 0.64% ID/g, small intestines 4.72 ± 0.55% ID/g, large intestines 4.13 ± 0.10%

radioactivity measurements of fecal matter (4–72 h: [64Cu]**1**: 3.03 ± 0.67 to 1.81 ± 0.74% ID/g;

<sup>64</sup>Cu]**2**: 9.32 ± 1.08 to 2.29 ± 0.53% ID/g; Figure 4). The GI uptake for [64Cu]**2** was more than double that of [64Cu]**1** at the earliest time point, but both peptides dropped over time to below 3.2% ID/g at 72 h. The liver uptake was moderate (<3% ID/g) throughout for both peptides; but it increased to significantly higher levels for the EB-ABM containing peptide

<sup>64</sup>Cu]**1,** beginning at 24 h, reaching >1.8-fold higher levels than [64Cu]**2** at 72 h (2.36 ± 0.51% ID/g vs. 1.30 ± 0.13% ID/g, respectively; *p* = 0.025, Figure 4). Overall, the EB-ABM containing peptide [64Cu]**1** had a less favorable pharmacokinetic profile with significantly higher uptake in the kidneys and liver, resulting in generally lower tumor-to-tissue ratios for

<sup>64</sup>Cu]**1** compared to [64Cu]**2**, most notably for the tumor-to-kidney ratio ([64Cu]**1** 0.13 ± 0.06/1 to 0.19 ± 0.08/1 vs. [64Cu]**2** 0.20 ± 0.06/1 to 0.44 ± 0.14/1), and the tumor-to-liver ratio ([64Cu]**1** 2.39 ± 0.59/1 to 1.47 ± 0.47/1 vs. [64Cu]**2** 2.72 ± 0.62/1 to 3.77 ± 0.72/1) (Figure S22).

both were cleared from the kidneys over time with accumulation dropping at 72 h ([64Cu]**1** 19.97 ± 6.91% ID/g; [64Cu]**2** 11.48 ± 1.02% ID/g; *p* = 0.103, Figure 4). Kidney accumulation for the ABM containing peptides [64Cu]**1** and [64Cu]**2** was initially higher than for the par-Integrin αvβ6-dependence of the tumor uptake was further substantiated by blocking studies with pre-administration of the respective nonradioactive peptide, which reduced tumor uptake to 2.91% ID/g and 2.89% ID/g for [64Cu]**1** and [64Cu]**2**, respectively (4 h; ∆ = −45% and −62%, *p* = 0.0124 and 0.0007, respectively; Figure S23).

<sup>64</sup>Cu]Cu DOTA-αvβ6-BP (20.37 ± 1.67% ID/g at 4 h to 6.81 ± 1.36%

<sup>64</sup>Cu]**1,** beginning at 24 h, reaching >1.8-fold higher levels than [64Cu]**2** at 72 h (2.36 ± 0.51% ID/g vs. 1.30 ± 0.13% ID/g, respectively; *p* = 0.025, Figure 4). Overall, the EB-ABM contain-

<sup>64</sup>Cu]**1** compared to [64Cu]**2**, most notably for the tumor-to-kidney ratio ([64Cu]**1** 0.13 ± 0.06/1 to 0.19 ± 0.08/1 vs. [64Cu]**2** 0.20 ± 0.06/1 to 0.44 ± 0.14/1), and the tumor-to-liver ratio ([64Cu]**1** 2.39 ± 0.59/1 to 1.47 ± 0.47/1 vs. [64Cu]**2** 2.72 ± 0.62/1 to 3.77 ± 0.72/1) (Figure S22).

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*3.6. Biodistribution*

ent non-ABM containing [

[

[

[

mulation by >3-to-4.5-fold compared to the [

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 13 of 20

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 13 of 20

**Figure 5.** (**A**) Biodistribution of select tissues at 4 h p.i. for [64Cu]**1**–**4**. (**B**) Tumor-to-organ ratios at 4 h p.i. for [64Cu]**1**–**4** [ <sup>64</sup>Cu]**1** (), [64Cu]**2** ( **Figure 3.** (**A**) Binding to human and mouse serum (*n* = 3/compound/condition; bars: SD). (**B**) Stability in human serum at 37 °C. (**C**) Stability in mouse serum at 37 °C for [<sup>64</sup>Cu]**1** (■) and [ <sup>64</sup>Cu]**2** (■). ), [64Cu]**3** ( **Figure 5.** (**A**) Biodistribution of select tissues at 4 h p.i. for [<sup>64</sup>Cu]**1**-**4**. (**B**) Tumor-to-organ ratios at 4 h p.i. for [<sup>64</sup>Cu]**1**-**4** [ <sup>64</sup>Cu]**1**(■), [ <sup>64</sup>Cu]**2** (■), [ <sup>64</sup>Cu]**3** (■), and [<sup>64</sup>Cu]**4** (■). ), and [64Cu]**4** ( **Figure 5.** (**A**) Biodistribution of select tissues at 4 h p.i. for [<sup>64</sup>Cu]**1**-**4**. (**B**) Tumor-to-organ ratios at 4 h p.i. for [<sup>64</sup>Cu]**1**-**4** [ <sup>64</sup>Cu]**1** (■), [ <sup>64</sup>Cu]**2** (■), [ <sup>64</sup>Cu]**3** (■), and [<sup>64</sup>Cu]**4** (■). ).

#### *3.8. PET Imaging*

The biodistributions for [64Cu]**1** and [64Cu]**2** in the BxPC-3 tumor model showed good tumor uptake (4 h to 72 h: [64Cu]**1** 5.29 ± 0.59 to 3.32 ± 0.46% ID/g, [64Cu]**2** 7.60 ± 0.43 to 4.91 ± 1.19% ID/g, Figure 4). Overall, tumor uptake of [64Cu]**2** appeared higher than of [64Cu]**1**, particularly at the earliest time point, and relative tumor washout over the total observed *3.7. Blocking Biodistribution* Integrin αvβ6-dependence of the tumor uptake was further substantiated by blocking studies with pre-administration of the respective nonradioactive peptide, which reduced tumor uptake to 2.91% ID/g and 2.89% ID/g for [ <sup>64</sup>Cu]**1** and [64Cu]**2**, respectively (4 h; Δ = −45% and −62%, *p* = 0.0124 and 0.0007, respectively; Figure S23). *3.7. Blocking Biodistribution* Integrin αvβ6-dependence of the tumor uptake was further substantiated by blocking studies with pre-administration of the respective nonradioactive peptide, which reduced tumor uptake to 2.91% ID/g and 2.89% ID/g for [ <sup>64</sup>Cu]**1** and [64Cu]**2**, respectively (4 h; Δ = −45% and −62%, *p* = 0.0124 and 0.0007, respectively; Figure S23). Overall, the BxPC-3 tumors were clearly visualized by PET imaging with both peptides at all time points (Figure 6); the PET imaging also showed that [64Cu]**2** provided the clearest images based on its superior tumor-to-background ratios. Most notably, as previously discussed for the biodistribution data, the PET images for [64Cu]**1** had much higher kidney accumulation and higher levels of radiation in the liver, indicative of possible in vivo instability of [64Cu]**1**, which had shown substantially higher degradation in mouse serum compared to [64Cu]**2**.

<sup>64</sup>Cu]**2**: 9.32 ± 1.08 to 2.29 ± 0.53% ID/g; Figure 4). The GI uptake for [64Cu]**2** was more than double that of [64Cu]**1** at the earliest time point, but both peptides dropped over time to below 3.2% ID/g at 72 h. The liver uptake was moderate (<3% ID/g) throughout for both peptides; but it increased to significantly higher levels for the EB-ABM containing peptide **Figure 6.** PET/CT imaging. Representative whole-body coronal maximum intensity projections (MIPs) of PET/CT images of mice bearing BxPC-3 xenograft tumors at 4 h, 24 h, 48 h, and 72 h p.i. of (**A**) [64Cu]**1** and (**B**) [64Cu]**2**. Arrow: tumor. Decay corrected PET data are shown in color, CT data in gray.

#### **4. Discussion**

Cancer remains a leading cause of death globally [48,49]. Many cancers exhibit high expression of the cell surface receptor integrin αvβ6, and expression levels correlate with poor prognosis and reduced progression-free and overall survival [31,32,38]. Therefore, integrin αvβ<sup>6</sup> has been identified as an important target both for imaging and treatment [50,51]. Receptor targeted delivery of radiopharmaceuticals is an important part of new approaches for improved cancer detection and therapy [48]. Peptides are attractive radiopharmaceuticals for both detection and treatment, because they are readily synthesized and can be chemically modified to optimize pharmacokinetics and metabolic stability. The addition of albumin binding moieties (ABMs) to numerous radiopharmaceuticals has demonstrated increased circulation time, reduced kidney uptake, and substantially increased tumor accumulation [18,52,53]. However, differences in the chemical structures of the ABM have been found at times to significantly affect the biodistribution, which ultimately determines target uptake, therapeutic efficacy, and off-target toxicity [52,54–56]. Thus, evaluation of different ABMs is important for optimal radiopharmaceutical performance towards the development of an αvβ6-targeted radiotherapeutic agent. Our laboratory continues to develop integrin αvβ6-targeting radiopharmaceuticals, including optimization of the core peptide structure [30] via PEGylation [14], and most recently the addition of an 4-(*p*iodophenyl)butyryl (IP) ABM, which has demonstrated improved accumulation in tumors for both the [18F]AlF NOTA and [64Cu]Cu DOTA radiolabeled IP-ABM-αvβ6-BP compared to the parent non-ABM αvβ6-BP [42,44]. To further evaluate the choice of preferred ABM for αvβ6-BP, the comparison of the IP-ABM with another prominent ABM, the Evans blue fragment (EB-ABM), was explored. The synthesis of both αvβ6-BP peptides containing different ABMs, [64Cu]**1** or [64Cu]**2** (Scheme 1), was done efficiently using a solid-phase approach, which allowed installation of the respective ABM-peptide from the same batch of peptidyl-resin by first coupling an orthogonally protected lysine allowing for the attachment of the DOTA-chelator at the *N*-terminus and either the EB-ABM **8** or the IP-ABM at the sidechain. The IP-ABM included an aspartate (D) residue as it is reported to result in better tumor retention [28]. After removal from the resin and purification, both DOTA-ABM-αvβ6-BP peptides (**1** and **2**) were efficiently radiolabeled with copper-64 to yield [ <sup>64</sup>Cu]**1** and [64Cu]**2** in high radiochemical purity >97%.

The ABM containing peptides [64Cu]**1** and [64Cu]**2** both demonstrated high tumor uptake at 4 h p.i., over 5% and 7.5% ID/g, respectively; representing a greater than 3 to-4.5-fold increase, respectively, from the non-ABM bearing [64Cu]Cu DOTA-αvβ6-BP (1.61 ± 0.70% ID/g) [44]. The improvement in tumor accumulation was greater for the IP-ABM peptide [64Cu]**2** than for the EB-ABM peptide [64Cu]**1**, and was in concordance with the cell binding to both DX3puroβ6 and BxPC-3 cells (Figure 2). Furthermore, the prolonged tumor uptake and retention (Figure 4A) were maintained for 72 h, and, in conjunction with rapid renal clearance, provided a high tumor-to-background ratio (Figure 5) and high contrast PET-images (Figure 6). Since the only difference between [64Cu]**1** and [64Cu]**2** is the ABM, and [64Cu]**2** showed significantly higher stability in serum compared to [64Cu]**1** (Figure 3), the observed differences in the tumor-to-background ratios could be attributed to the improved stability. This study adds to the growing number of literature reports describing improved tumor uptake following the incorporation of ABMs [4,7,11]. For example, the small molecule PSMA-617, a radiopharmaceutical targeting the prostate specific membrane antigen (PSMA), exhibited approximately a fivefold increase in tumor accumulation with the addition of an EB-ABM at 4 h and a twofold increase for the IP-ABM modified PSMA-617, compared to the unmodified (non-ABM bearing) PSMA-617; furthermore, the EB-ABM PSMA-617 maintained tumor accumulation over time (65.6–77.3% ID/g from 4 h to 48 h) [55]. In another study with PSMA-617, the addition of the IP-ABM also resulted in twofold higher accumulation in tumor tissue as compared to the non-ABM containing PSMA-617 agent (non-ABM PSMA-617: 38% ID/g vs. IP-ABM PSMA-617: 75.7% ID/g at 24 h) [28,57]. Other small molecule PSMA agents modified with ABMs have also shown improvements in tumor accumulation, with the EB-ABM MCG

PSMA agent having around a fourfold increase in tumor accumulation (MCG non-ABM: 10.9% ID/g vs. MCG-ABM: 40.4% ID/g at 24 h) [53] and an IP-ABM PSMA agent CTT1403 exhibiting >18-fold improvement in tumor accumulation (CTT1401 non-ABM: 2.2% ID/g vs. CTT1403-ABM: 40% ID/g at 24 h [54]. The addition of ABMs to other small molecule radiopharmaceuticals has also been shown to improve tumor accumulation with the small molecule radioligand folic acid modified with the IP-ABM having a threefold increase in tumor accumulation (ABM: 19.5% ID/g vs. non-ABM: 7% ID/g at 24 h, p.i.) with a considerably lower kidney accumulation (ABM: 28% ID/g vs. non-ABM: 70% ID/g at 4 h) [52,58].

Aside from small molecule radiopharmaceuticals, substantial benefits from the addition of ABMs to peptide radiopharmaceuticals have been shown; for example, the large peptide exendin-4 (39 amino acids), which targets the glucagon-like peptide 1 (GLP-1) receptor, when modified with the IP-ABM, demonstrated an improved stability and a twofold increase in tumor accumulation at 4 h, along with reduced kidney retention by more than half [7,59]. The small five amino acid integrin αvβ<sup>3</sup> targeting cyclic peptide (cRGDfK) modified with EB-ABM and radiolabeled as [64Cu]Cu NOTA-EB-cRGDfK displayed a >16-fold improvement (vs. [64Cu]Cu NOTA-cRGDfK) in tumor accumulation in a U87MG glioblastoma tumor model (with ABM: 16.6% ID/g vs. non-ABM: <1.1% ID/g), but only had about a fivefold improvement in MDA-MB-435 melanoma and HT29 colorectal adenocarcinoma models [18]. The somatostatin receptor targeting peptide octreotide (TATE), which is eight amino acids in size, has seen some of the greatest improvements in tumor accumulation upon modification with an ABM. For example, the EB-ABM modified [ <sup>177</sup>Lu]Lu DOTA-EB-TATE provided a greater than eightfold increase in the tumor accumulation at 24 h (with ABM: 78.8% ID/g vs. non-ABM: 9.3% ID/g, respectively) [60] and the [86Y]Y DOTA-EB-TATE showed a larger enhancement with a between 30- and 60-fold increase in tumor accumulation compared to [86Y]Y DOTA-TATE, depending on the tumor model [6]. These studies paved the way for clinical trials where [177Lu]Lu DOTA-EB-TATE showed an extended circulation which led to a 7.9-fold increase in tumor dose delivery [61]. Overall, these studies illustrate the potential benefits of including an ABM on targeted peptide receptor radionuclide therapy (PRRT).

The addition of either EB-ABM or the IP-ABM on the αvβ6-BP did significantly increase tumor accumulation (three-to-fivefold from the non-ABM-αvβ6-BP) and the overall clearance properties of the ABM-modified αvβ6-BP peptides [64Cu]**1** and [64Cu]**2** were similar with predominantly renal excretion. The organ with the highest accumulation was the kidneys, with the initial kidney uptake of the EB-ABM peptide [64Cu]**1** having more than double that of the IP-ABM peptide [64Cu]**<sup>2</sup>** (4 h: 75.5 <sup>±</sup> 7.3% ID/g vs. 33.6 <sup>±</sup> 5.4% ID/g, *p* = 0.0013), with both dropping to approximately one third of their initial value at 72 h p.i. (20.0 ± 6.9% ID/g and 11.4 ± 1.0% ID/g, respectively, *p* = 0.10, Figure 4). The introduction of the IP-ABM to the αvβ6-BP significantly reduced kidney accumulation, which we hypothesize is due to the higher stability of the IP-ABM [64Cu]**2** over the EB-ABM [64Cu]**1**. These data are promising and indicate that renal toxicity would be less of a concern for PRRT of αvβ6-BP agents using the IP-ABM. The observed effects of the different ABMs on kidney uptake and retention are comparable to other radiopharmaceutical ABMadducts, for example, the ABM modified peptide [177Lu]Lu DOTA-TATE showed that the IP-ABM-analogue also provided lower kidney accumulation that was more rapidly cleared (dropping from ~20% ID/g at 4 h to ~5% ID/g at 72 h) compared to the EB-ABM-analogue (~30% ID/g at 4 h to ~15% ID/g at 72 h) [29,60]. This similar kidney accumulation and retention trend was also observed with the small molecule PSMA-617 agent, where the EB-PSMA-617 had considerably higher kidney accumulation and retention compared to the IP-PSMA-617, which had rapid kidney clearance (EB-PSMA-617: >20% ID/g at 4 h, which remained at 48 h vs. IP-ABM-PSMA-617: ~10% ID/g at 4 h dropping to <5% ID/g at 48 h) [55]. Both [64Cu]**1** and [64Cu]**2** also displayed some secondary clearance through the gastrointestinal (GI) tract and excretion of radioactivity in the feces (Figures S20 and S21). The IP-ABM modified peptide [64Cu]**2** had higher GI accumulation, with the highest

uptake in the stomach of 18.1 ± 2.9% ID/g at 4 h, though, gratifyingly, both peptide's GI accumulation dropped down to less than one-fifth of their respective original value (≤3.2% ID/g at 72 h, Figure 4).

The non-αvβ6-targeting ABM controls [64Cu]**3** and [64Cu]**4** were used to evaluate non-specific uptake and demonstrate that the enhanced tumor accumulation of [64Cu]**1** or [ <sup>64</sup>Cu]**2** resulted from integrin αvβ<sup>6</sup> receptor mediated uptake, as opposed to the enhanced permeability and retention (EPR) effect. As expected, [64Cu]**3** and [64Cu]**4** largely remained in the blood, thus mostly acting as blood pool imaging agents with high blood accumulation of 39.0% ID/g and 9.5% ID/g, respectively, at 4 h (Figure S25) and mirrored other similar non-targeted ABMs, such as the EB-ABM compound [64Cu]Cu NOTA-EB (NEB, ~15% ID/g at 4 h, dropping to ~10% ID/g at 1 d) [16,23]. Compared to the ABM peptides [64Cu]**1** and [64Cu]**2**, accumulation of [64Cu]**3** and [64Cu]**4** generally increased in organs with high blood flow (viz. heart, liver, and lungs; Figure 5A) but was lower in the kidneys (though the EB compound was still higher than the IP compound with 18.6% ID/g and 4.3% ID/g, respectively, at 4 h; Figure 5A), highlighting the effect of both the properties of the ABM as well as the targeting peptide moiety on kidney uptake. Both non-targeted [64Cu]**3** and [ <sup>64</sup>Cu]**4**, due to their much higher blood accumulation (>9–39-fold higher than [64Cu]**1** and [ <sup>64</sup>Cu]**2**) and longer blood residence time, provided much higher tumor accumulation at 4 h than the two peptides [64Cu]**1** and [64Cu]**2** (Figure 5A). However, the non-targeted [ <sup>64</sup>Cu]**3** and [64Cu]**4** showed minimal binding (<4.3%) in cell binding studies to both the αvβ6-expressing and αvβ6-null cells (Figure S24), thus their higher tumor accumulation compared to [64Cu]**1** and [64Cu]**2** was attributed to the EPR effect (which, together with the long circulation, resulted in the expectedly low tumor-to-blood ratios of <0.9/1 (Figure 5B, Figure S26). By comparison, [64Cu]**1** and [64Cu]**2** showed high and αvβ6-dependent cell binding (>30–60% binding; ~20:1 for DX3puroβ6 (+)/DX3puro (−) cells), and in vivo tumor uptake was efficiently blocked by the pre-administration of metal free **1** and **2**, respectively, supporting integrin αvβ6-dependent tumor accumulation (Figure S23). Taken together, the tumor uptake observed for the integrin αvβ6-binding peptides [64Cu]**1** and [64Cu]**2** was attributed to specific targeting of the integrin αvβ<sup>6</sup> receptor. Both ABM modified αvβ6-BP peptides had improved pharmacokinetic profiles from the parent peptide and overall [64Cu]**2** demonstrated a more favorable biodistribution. Tumor retention of [64Cu]**1** and [64Cu]**2** was good over the three day study period, with each retaining about two-thirds of the original (4 h) uptake at 72 h p.i. The PET image quality improved, most notably for [ <sup>64</sup>Cu]**2** over time after the initial uptake period (i.e., after 24 h p.i.) as a result of faster washout from non-target tissues (Figure 6). The high absolute tumor uptake of [64Cu]**2**, its efficient binding and internalization to αvβ6-expressing cells (Figure 2), and its better serum stability (Figure 3) demonstrate the potential of using the [64Cu]**2** as an integrin αvβ6-targeted peptide receptor radionuclide therapy (PRRT) agent where the copper-64 is replaced by a therapeutic radioisotope such as lutetium-177.

## **5. Conclusions**

The effect of Evans blue (EB) and 4-(*p*-iodophenyl)butyryl (IP)-based albumin binding moieties (ABMs) on the pharmacokinetics of αvβ6-BP, a peptide targeting the cancerassociated cell surface receptor integrin αvβ<sup>6</sup> was investigated. The albumin binding moieties on αvβ6-BP did not interfere with integrin αvβ<sup>6</sup> affinity or selectivity in vitro. In vivo in a BxPC-3 pancreatic tumor xenograft mouse model, the IP-ABM-modified αvβ6- BP [64Cu]**2** had a considerably more favorable pharmacokinetic profile compared to the EB-ABM-modified αvβ6-BP [64Cu]**1**, with higher tumor uptake, reduced kidney and liver uptake, and improved tumor-to-background ratios that led to a clearer tumor visualization by PET imaging. Furthermore, the IP-ABM-modified αvβ6-BP [64Cu]**2** had superior serum stability, making it a lead candidate for future integrin αvβ6-targeted imaging and therapy studies.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics14040745/s1, S1–S26. Table S1–S3: Table of Contents, Table S3: RP-HPLC methods, Figure S4: Schematic for solid phase reaction of EB-ABM **8** to peptidyl resin of DOTA-K(NH<sup>2</sup> -αvβ<sup>6</sup> -BP to produce DOTA-EB-αvβ<sup>6</sup> -BP **1** after cleavage and pictorial of the reaction of **8** with peptidyl resin of DOTA-K(NH<sup>2</sup> )-αvβ<sup>6</sup> -BP, Figure S5: RP-HPLC and MALDI-TOF of DOTA-EB-αvβ<sup>6</sup> - BP **1**, Figure S6: Radio-RP-HPLC of [64Cu]**1** and co-injection radio-RP-HPLC of [NatCu]**1** and [64Cu]**1**, Figure S7: MALDI-TOF of [NatCu]**1**, Figure S8: RP-HPLC and MALDI-TOF of DOTA-IP-αvβ<sup>6</sup> -BP **2**, Figure S9: Radio-RP-HPLC of [64Cu]**2** and co-injection radio-RP-HPLC of [NatCu]**2** and [64Cu]**2**, Figure S10: MALDI-TOF of [NatCu]**2**, Figure S11: RP-HPLC and MALDI-TOF of DOTA-EB-ABM **3**, Figure S12: Radio-RP-HPLC of [64Cu]**3** and co-injection radio-RP-HPLC of [NatCu]**3** and [64Cu]**3**, Figure S13: MALDI-TOF of [NatCu]**3**, Figure S14: RP-HPLC and MALDI-TOF of DOTA-IP-ABM **4**, Figure S15: Radio-RP-HPLC of [64Cu]**4** and co-injection radio-RP-HPLC of [NatCu]**4** and [64Cu]**4**, Figure S16: MALDI-TOF of [NatCu]**4**, Figure S17: RP-HPLC and ESI-FTMS of compound **6**, Figure S18: RP-HPLC and ESI-FTMS of EB-ABM **8**, Figure S19: <sup>1</sup>H NMR and COSY of EB-ABM **8**, Figure S20: Biodistribution of [64Cu]**1**, Figure S21: Biodistribution of [64Cu]**2**, Figure S22: Tumor-to-organ ratios from 4 h to 72 h p.i. of [64Cu]**1** and [64Cu]**2**, Figure S23: Blocking biodistribution of [64Cu]**1** and [ <sup>64</sup>Cu]**2**, Figure S24: Cell binding assay for [64Cu]**3** and [64Cu]**4**, Figure S25: Biodistribution of [64Cu]**3** and [64Cu]**4**, Figure S26: Summary of Tumor-to-organ ratios at 4 h for [64Cu]**1**–**4**.

**Author Contributions:** Conceptualization, J.L.S. and R.A.D.; methodology, R.A.D. and S.H.H.; formal analysis, J.L.S., R.A.D. and S.H.H.; synthesis and radiolabeling with copper-64, R.A.D.; compound characterization and purification and formulation, R.A.D.; serum stability assay, R.A.D.; cell culture, R.H.; cell binding assay and serum binding assays, S.H.H. and R.A.D.; biodistribution, S.H.H., R.H. and R.A.D.; resources, J.L.S.; data curation, R.A.D.; writing—original draft preparation, R.A.D.; writing—review and editing, R.A.D., J.L.S. and S.H.H.; supervision, J.L.S.; funding acquisition, J.L.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Institutes of Health's National Cancer Institute, grants number R01CA199725 and R50CA211556-01.

**Institutional Review Board Statement:** Radioactive work was conducted under radioactive use authorization 9098 managed by University of California, Davis Radiation Safety Services. All animal and biological research were conducted under biological use authorization R1580 and all animal work was conducted in accordance with procedures pre-approved by the Institutional of Animal Care and Use Committee (IACUC) at the University of California, Davis which is regulated by several independent resources. Accreditation and oversight has been approved since 1966 by AAALAC #000029 and by the Office of Laboratory Animal Welfare (OLAW) #D16-00272 (A3433-01).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Additional data supporting the reported results can be found in the Supplementary Materials (S1–S26).

**Acknowledgments:** We would like to thank the Center for Molecular and Genomic Imaging at UC Davis, Charles Smith and Sarah Tam for their technical support of injections during animal studies and running of the PET/CT scanners.

**Conflicts of Interest:** The authors declare the following competing financial interest(s): S. H. Hausner is a co-inventor of intellectual property related to αvβ<sup>6</sup> -BP. J. L. Sutcliffe is founder and CEO of and holds ownership interest (including patents) in Luminance Biosciences, Inc., and is a co-inventor of intellectual property related to αvβ<sup>6</sup> -BP. The funding agencies had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


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