Preclinical Characterization of DPI-4452: A 68Ga/177Lu Theranostic Ligand for Carbonic Anhydrase IX

Abstract

The membrane protein carbonic anhydrase IX (CAIX) is highly expressed in many hypoxic or von Hippel-Lindau tumor suppressor-mutated tumor types. Its restricted expression in healthy tissues makes CAIX an attractive diagnostic and therapeutic target. DPI-4452 is a CAIX-targeting cyclic peptide with a DOTA cage, allowing radionuclide chelation for theranostic purposes. Here, we report CAIX expression in multiple tumor types and provide in vitro and in vivo evaluations of ⁶⁸Ga-labeled DPI-4452 ([⁶⁸Ga]Ga-DPI-4452) and ¹⁷⁷Lu-labeled DPI-4452 ([¹⁷⁷Lu]Lu-DPI-4452). Methods: CAIX expression was assessed by immunohistochemistry with a panel of tumor and healthy tissues. The molecular interactions of complexed and uncomplexed DPI-4452 with CAIX were assessed by surface plasmon resonance and cell-binding assays. In vivo characterization of radiolabeled and nonradiolabeled DPI-4452 was performed in HT-29 colorectal cancer (CRC) and SK-RC-52 clear cell renal cell carcinoma (ccRCC) human xenograft mouse models and in healthy beagle dogs. Results: Overexpression of CAIX was shown in several tumor types, including ccRCC, CRC, and pancreatic ductal adenocarcinoma. DPI-4452 specifically and selectively bound CAIX with subnanomolar affinity. In cell-binding assays, DPI-4452 displayed comparably high affinities for human and canine CAIX but a much lower affinity for murine CAIX, demonstrating that the dog is a relevant species for biodistribution studies. DPI-4452 was rapidly eliminated from the systemic circulation of beagle dogs. The highest uptake of [⁶⁸Ga]Ga-DPI-4452 and [¹⁷⁷Lu]Lu-DPI-4452 was observed in the small intestine and stomach, 2 organs known to express CAIX. Uptake in other organs (e.g., kidneys) was remarkably low. In HT-29 and SK-RC-52 xenograft mouse models, both [⁶⁸Ga]Ga-DPI-4452 and [¹⁷⁷Lu]Lu-DPI-4452 showed tumor-selective uptake; in addition, [¹⁷⁷Lu]Lu-DPI-4452 significantly reduced tumor growth. These results demonstrated the theranostic potential of DPI-4452. Conclusion: DPI-4452 selectively targets CAIX. [⁶⁸Ga]Ga-DPI-4452 and [¹⁷⁷Lu]Lu-DPI-4452 localized to tumors and were well tolerated in mice. [¹⁷⁷Lu]Lu-DPI-4452 demonstrated strong tumor growth inhibition in 2 xenograft mouse models. Thus, the 2 agents potentially provide a theranostic approach for selecting and treating patients with CAIX-expressing tumors such as ccRCC, CRC, and pancreatic ductal adenocarcinoma.

Keywords: 177Lu; 68Ga; CAIX; dog; mouse.

Graphical abstract

FIGURE 1.

[¹¹¹In]In-DPI-4452 tissue pharmacokinetics in mice bearing HT-29 and SK-RC-52 xenografts. Mean uptake (radioactivity concentration as percentage injected dose per gram of tissue [%ID/g]) over time in key organs after injection of 30 MBq of [¹¹¹In]In-DPI-4452 into mice with human CRC HT-29 (A) and human ccRCC SK-RC-52 (B) subcutaneous xenografts. Shown are measurements for individual mice. Line represents mean. n = 3.

FIGURE 2.

[⁶⁸Ga]Ga-DPI-4452 and [¹⁷⁷Lu]Lu-DPI-4452 biodistribution in mice with HT-29 and SK-RC-52 xenografts. Shown are maximum-intensity projection (MIP) images of representative mice bearing subcutaneous xenografts of HT-29 (A) or SK-RC-52 (B) tumors. Each mouse was imaged by PET/CT at 1 h after injection (p.i.) with 9 MBq of [⁶⁸Ga]Ga-DPI-4452 per animal (left) and, 7 d later, by SPECT/CT at 4 h after injection with 33 MBq of [¹⁷⁷Lu]Lu-DPI-4452 per animal (right). Scale bar represents radioactivity concentration as percentage injected dose per gram of tissue (%ID/g) (values in parentheses). B = bladder; K = kidney; T = tumor.

FIGURE 3.

Uptake and treatment effect of [¹⁷⁷Lu]Lu-DPI-4452 in subcutaneous HT-29 and SK-RC-52 human xenograft mouse models. (A and B) Growth of HT-29 (A) and SK-RC-52 (B) xenografts treated with single doses of vehicle, 100 or 33 MBq of [¹⁷⁷Lu]Lu-DPI-4452, or 3 once-weekly (QW × 3) 33 MBq doses of [¹⁷⁷Lu]Lu-DPI-4452. Arrowheads indicate days of treatment (n = 10 per group). (C and D) Tumor uptake (percentage injected dose per gram of tissue, %ID/g) for HT-29 (C) and SK-RC-52 (D) xenografts at 4 h after injection (n = 3 per group). Shown are mean (bar) and individual values.

FIGURE 4.

Comparison of [⁶⁸Ga]Ga-DPI-4452 and [¹⁷⁷Lu]Lu-DPI-4452 biodistributions in healthy dogs. PET/CT images taken at 1 h after injection (p.i.) of [⁶⁸Ga]Ga-DPI-4452 at 200 MBq (left) and SPECT/CT images taken at 1 h after injection of [¹⁷⁷Lu]Lu-DPI-4452 at 1000 MBq (right) for one female beagle dog (with 2-wk interval between 2 injections). Scale bars represent SUV.

FIGURE 5.

[¹⁷⁷Lu]Lu-DPI-4452 tissue distribution in healthy dogs. [¹⁷⁷Lu]Lu-DPI-4452 organ uptake (radioactivity concentration) for female and male beagle dogs at 1 h (A), 6 h (B), 24 h (C), and 48 h (D) after injection of 1000 MBq. Uptake (radioactivity concentration) is presented as percentage injected dose per gram of tissue (%ID/g). n = 3.

FIGURE 6.

[¹¹¹In]In-DPI-4452 plasma pharmacokinetics in healthy dogs. Shown is mean plasma radioactivity concentration (percentage injected dose per gram [%ID/g]) after injection of [¹¹¹In]In-DPI-4452 at 25 MBq/kg with variable specific activities. Shown are individual measurements. Line represents mean. n = 2.