[111In]In/[177Lu]Lu-AAZTA5-LM4 SST2R-Antagonists in Cancer Theranostics: From Preclinical Testing to First Patient Results

Abstract

Aiming to expand the application of the SST₂R-antagonist LM4 (DPhe-c[DCys-4Pal-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH₂) beyond [⁶⁸Ga]Ga-DATA^5m-LM4 PET/CT (DATA^5m, (6-pentanoic acid)-6-(amino)methy-1,4-diazepinetriacetate), we now introduce AAZTA⁵-LM4 (AAZTA⁵, 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-[pentanoic-acid]perhydro-1,4-diazepine), allowing for the convenient coordination of trivalent radiometals of clinical interest, such as In-111 (for SPECT/CT) or Lu-177 (for radionuclide therapy). After labeling, the preclinical profiles of [¹¹¹In]In-AAZTA⁵-LM4 and [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 were compared in HEK293-SST₂R cells and double HEK293-SST₂R/wtHEK293 tumor-bearing mice using [¹¹¹In]In-DOTA-LM3 and [¹⁷⁷Lu]Lu-DOTA-LM3 as references. The biodistribution of [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 was additionally studied for the first time in a NET patient. Both [¹¹¹In]In-AAZTA⁵-LM4 and [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 displayed high and selective targeting of the HEK293-SST₂R tumors in mice and fast background clearance via the kidneys and the urinary system. This pattern was reproduced for [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 in the patient according to SPECT/CT results in a monitoring time span of 4-72 h pi. In view of the above, we may conclude that [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 shows promise as a therapeutic radiopharmaceutical candidate for SST₂R-expressing human NETs, based on previous [⁶⁸Ga]Ga-DATA^5m-LM4 PET/CT, but further studies are needed to fully assess its clinical value. Furthermore, [¹¹¹In]In-AAZTA⁵-LM4 SPECT/CT may represent a legitimate alternative diagnostic option in cases where PET/CT is not available.

Keywords: AAZTA5 chelator; AAZTA5-LM4; In-111; Lu-177; SPECT imaging; neuroendocrine tumors; radionuclide therapy; somatostatin subtype 2 receptor (SST2R)-antagonist; theranostics.

Figure 1

Chemical structures of (a) AAZTA⁵-LM4; (b) DOTA-LM3; the two analogs differ in the chelator (AAZTA⁵: 1,4-bis(carboxymethyl)-6-[bis (carboxymethyl)]amino-6-[pentanoic-acid]perhydro-1,4-diazepine and DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and in the residue at position 3: 4Pal (4-pyridyl)alanine) in LM4 and Tyr in LM3; DAph(Cbm): D-4-(carbamoyl)amino-phenylalanine).

Figure 2

Displacement of [¹²⁵Tyr²⁵]LTT-SS28 from SST₂R binding sites in HEK293-SST₂R cell membranes by increasing concentrations of ○ AAZTA⁵-LM4 (IC₅₀ = 1.69 ± 0.47 nM, n = 4); ● [^natIn]In-AAZTA⁵-LM4 (IC₅₀ = 0.45 ± 0.05 nM, n = 3) and ● [^natLu]Lu-AAZTA⁵-LM4 (IC₅₀ = 0.55 ± 0.38 nM, n = 3); results represent mean IC₅₀ values ± sd, n: number of separate experiments in triplicate.

Figure 3

Radioligand uptake internalization during 1 h incubation at 37 °C in HEK293-SST₂R cells. (a) ■, [¹¹¹In]In-AAZTA⁵-LM4 and ■, [¹¹¹In]In-DOTA-LM3; (b) ■, [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 and ■, [¹⁷⁷Lu]Lu-DOTA-LM3; MB = specific membrane bound, Internalized = specific internalized; Total = specific MB + specific Internalized, NS MB = non-specific MB, NS Intern = non-specific internalized. Results were acquired from 3 independent experiments performed in triplicate.

Figure 4

Comparative biodistribution data in SCID mice bearing twin HEK293-SST₂R and wtHEK293 xenografts. (a) ■, [¹¹¹In]In-AAZTA⁵-LM4 and ■, [¹¹¹In]In-DOTA-LM3; (b) ■, [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 and ■, [¹⁷⁷Lu]Lu-DOTA-LM3; solid bars for 4 h pi, checkered bars for 24 h pi and crossed bars for 48 h pi. Data are expressed as %IA/g and shown as average values ± sd, n = 4; Bl: blood, Li: liver, He: heart, Ki: kidneys, St: stomach, In: intestines, Sp: spleen, Mu: muscle, Lu: lungs, Pa: pancreas, Fe: femur, Tu+: HEK293-SST₂R tumor, Tu-: wtHEK293 tumor.

Figure 5

Static whole-body SPECT/CT images of SCID mice bearing twin HEK293-SST₂R and wtHEK293 tumors in their flanks at (a) and (b) 4 h pi and (c) at 24 h pi of [¹¹¹In]In-AAZTA⁵-LM4; orange arrows are pointing to HEK293-SST₂R xenografts and light blue arrows to wtHEK293 tumors, devoid of SST₂R expression. Intense uptake is observed in the SST₂R-expressing tumors but no uptake is evident in the negative controls. Yellow arrows are directed toward the kidneys; the initial kidney uptake at (a) and (b) 4 h pi notably declines at (c) 24 h pi. The color bars indicate the difference in accumulated activity (purple being the lowest and white the highest level of accumulation).

Figure 6

Correlation of PET/CT imaging (a–d) with SPECT/CT imaging (e–i) in a 68-year-old female patient with a stomach NET with multiple lymph node and liver metastases. (a) Mild to negligible radiotracer concentration in pathological lesions on PET/CT with [⁶⁸Ga]Ga-DOTA-NOC at 1 h pi (reference) vs. PET/CT with [⁶⁸Ga]Ga-DATA^5m-LM4 at (b) 10 min pi, (c) 1 h pi and (d) 3 h pi, including whole-body and transaxial views. As indicated by the red arrows, mild to negligible uptake was displayed by [⁶⁸Ga]Ga-DOTA-NOC in the liver metastases, whereas the uptake of [⁶⁸Ga]Ga-DATA^5m-LM4 was notably superior. The same patient was selected for sequential SPECT/CT imaging with [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 at (e) 4 h, (f) 17 h, (g) 24 h, (h) 48 h and (i) 72 h pi (anterior/posterior views included for each time point).