Comparative biodistribution of NODAGA-Z_HER2:S1, NOTA-Z_HER2:S1 and DOTA-Z_HER2:S1 labelled with gallium-68 and indium-111 after intravenous injection in female BALB/C nu/nu mice bearing SKOV-3 xenografts.

<p>Data are presented as an average % IA/g and standard deviation for four mice. Data concerning bone from one mice injected with ⁶⁸Ga-NOTA-Z_HER2:S1 and bone and muscle form one mice injected with ⁶⁸Ga-NODAGA-Z_HER2:S1were excluded due to low counts.</p>a<p>significant difference (p<0.05) between ⁶⁸Ga-DOTA-Z_HER2:S1 and ¹¹¹In-DOTA-Z_HER2:S1.</p>b<p>significant difference (p<0.05) between ⁶⁸Ga-NOTA-Z_HER2:S1 and ¹¹¹In-NOTA-Z_HER2:S1.</p>c<p>significant difference (p<0.05) between ⁶⁸Ga-NODAGA-Z_HER2:S1 and ¹¹¹In-NODAGA-Z_HER2:S1.</p>d<p>significant difference (p<0.05) between ⁶⁸Ga-DOTA-Z_HER2:S1 and ⁶⁸Ga-NOTA-Z_HER2:S1.</p>e<p>significant difference (p<0.05) between ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-NODAGA-Z_HER2:S1.</p>f<p>significant difference (p<0.05) between ⁶⁸Ga-NODAGA-Z_HER2:S1 and ⁶⁸Ga-DOTA-Z_HER2:S1.</p>*<p>Data for gastrointestinal (GI) tract and carcass are presented as %IA per whole organ.</p

Labeling yield (decay corrected), radiochemical purity and stability of ⁶⁸Ga-labeled Affibody molecules.

*<p>To evaluate stability, conjugates were incubated with 500-fold excess of EDTA for 1 h and then analyzed using ITLC. Control samples were incubated in PBS. The experiments were performed in duplicates. The data present an average conjugate-associated radioactivity and maximum error.</p

Tumor-to-organ ration data 2h after injection for NODAGA-Z_HER2:S1, NOTA-Z_HER2:S1 and DOTA-Z_HER2:S1 in mice bearing SKOV-3 xenografts.

<p>Data are presented as an average and standard deviation for four mice. Data concerning bone from one mice injected with ⁶⁸Ga-NOTA-ZHER2:S1 and bone and muscle form one mice injected with ⁶⁸Ga-NODAGA-ZHER2:S1were excluded due to low counts.</p>a<p>significant difference (p<0.05) between ⁶⁸Ga-DOTA-Z_HER2:S1 and ¹¹¹In-DOTA-Z_HER2:S1.</p>b<p>significant difference (p<0.05) between ⁶⁸Ga-NOTA-Z_HER2:S1 and ¹¹¹In-NOTA-Z_HER2:S1.</p>c<p>significant difference (p<0.05) between ⁶⁸Ga-NODAGA-Z_HER2:S1 and ¹¹¹In-NODAGA-Z_HER2:S1.</p>d<p>significant difference (p<0.05) between ⁶⁸Ga-DOTA-Z_HER2:S1 and ⁶⁸Ga-NOTA-Z_HER2:S1.</p>e<p>significant difference (p<0.05) between ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-NODAGA-Z_HER2:S1.</p>f<p>significant difference (p<0.05) between ⁶⁸Ga-NODAGA-Z_HER2:S1 and ⁶⁸Ga-DOTA-Z_HER2:S1.</p

MicroPET/CT imaging of HER2 expression in SKOV3 xenografts in BALB/C nu/nu mice using ⁶⁸Ga-NODAGA-Z_HER2:S1.

<p>The image was acquired 2 h after administration of the tracer. Arrows point at tumor (T) and kidneys (K).</p

Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of ¹¹¹In-Labeled Synthetic Anti-HER2 Affibody Molecules

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with ^99mTc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z_HER2:S1 at three different positions: DOTA-A1-Z_HER2:S1 (N-terminus), DOTA-K58-Z_HER2:S1 (C-terminus), and DOTA-K50-Z_HER2:S1 (middle of helix 3). The affinity for HER2 differed slightly among the variants and the <i>K</i>_D values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z_HER2:S1, DOTA-K50-Z_HER2:S1, and DOTA-K58-Z_HER2:S1, respectively. Z_HER2:S1-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z_HER2:S1 (64 °C) and DOTA-K58-Z_HER2:S1 (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with ¹¹¹In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z_HER2:S1and DOTA-K58-Z_HER2:S1 showed the highest uptake. Overall, DOTA-K58-Z_HER2:S1 provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization

Biophysical characteristics of the Affibody conjugates [22].

<p>Biophysical characteristics of the Affibody conjugates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070028#pone.0070028-Malmberg2" target="_blank">[22]</a>.</p

Specificity of targeting of SKOV-3 xenografts in BALB/C nu/nu mice using ⁶⁸Ga-DOTA-Z_HER2:S1, ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-NODAGA-Z_HER2:S1 at 1h after injection.

<p>The total injected radiolabeled peptide dose per mouse was 3 µg (0.42 nmol). The blocked group was subcutaneously pre-injected with an excess amount (1000 µg, 150 nmol) of non-labelled Z_HER2∶342 to saturate binding sites of HER2. Results are expressed as percentage of injected activity per gram of tissue (%IA/g) and presented as mean values for four mice and standard deviations. Uptake of radioactivity was significant lower (p<0.0005) for pre-saturated SKOV-3 tumors.</p

Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins

Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with ^99mTc­(CO)₃. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)₃-tag instead of the commonly used hexahistidine (H₆)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H₆- or (HE)₃-tags in the N-termini were prepared. The constructs, (HE)₃-ADAPT6 and H₆-ADAPT6, were labeled with two different nuclides, ^99mTc or ¹¹¹In. The labeling with ^99mTc­(CO)₃ utilized the histidine-containing tags, while ¹¹¹In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For ¹¹¹In-labeled ADAPTs, the use of (HE)₃ provided a significantly (<i>p</i> < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of ¹¹¹In-(HE)₃-ADAPT6 and H₆-ADAPT6 at later time points. Interestingly, in the case of ^99mTc, ^99mTc­(CO)₃-H₆-ADAPT6 provided significantly (<i>p</i> < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP

Cellular processing of ⁶⁸Ga-NODAGA-Z_HER2:S1, ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-DOTA-Z_HER2:S1 by HER2-expressing cells SKOV-3 in vitro.

<p>Cells were incubated with labeled compound at 37°C. Cell bound activity is normalized to the maximum uptake. Data are presented as mean values for three cell dishes and standard deviations. Error bars might be smaller than the symbols.</p

DataSheet_1_177Lu-labeled PSMA targeting therapeutic with optimized linker for treatment of disseminated prostate cancer; evaluation of biodistribution and dosimetry.docx

IntroductionProstate specific membrane antigen (PSMA), highly expressed in metastatic castration-resistant prostate cancer (mCRPC), is an established therapeutic target. Theranostic PSMA-targeting agents are widely used in patient management and has shown improved outcomes for mCRPC patients. Earlier, we optimized a urea-based probe for radionuclide visualization of PSMA-expression in vivo using computer modeling. With the purpose to develop a targeting agent equally suitable for radionuclide imaging and therapy, the agent containing DOTA chelator was designed (BQ7876). The aim of the study was to test the hypothesis that 177Lu-labeled BQ7876 possesses target binding and biodistribution properties potentially enabling its use for radiotherapy. MethodsBQ7876 was synthesized and labeled with Lu-177. Specificity and affinity of [177Lu]Lu-BQ7876 to PSMA-expressing PC3-pip cells was evaluated and its processing after binding to cells was studied. Animal studies in mice were performed to assess its biodistribution in vivo, target specificity and dosimetry. [177Lu]Lu-PSMA-617 was simultaneously evaluated for comparison.ResultsBQ7876 was labeled with Lu-177 with radiochemical yield >99%. Its binding to PSMA was specific in vitro and in vivo when tested in antigen saturation conditions as well as in PSMA-negative PC-3 tumors. The binding of [177Lu]Lu-BQ7876 to living cells was characterized by rapid association, while the dissociation included a rapid and a slow phase with affinities KD1 = 3.8 nM and KD2 = 25 nM. The half-maximal inhibitory concentration for natLu-BQ7876 was 59 nM that is equal to 61 nM for natLu-PSMA-617. Cellular processing of [177Lu]Lu-BQ7876 was accompanied by slow internalization. [177Lu]Lu-BQ7876 was cleared from blood and normal tissues rapidly. Initial elevated uptake in kidneys decreased rapidly, and by 3 h post injection, the renal uptake (13 ± 3%ID/g) did not differ significantly from tumor uptake (9 ± 3%ID/g). Tumor uptake was stable between 1 and 3 h followed by a slow decline. The highest absorbed dose was in kidneys, followed by organs and tissues in abdomen.DiscussionBiodistribution studies in mice demonstrated that targeting properties of [177Lu]Lu-BQ7876 are not inferior to properties of [177Lu]Lu-PSMA-617, but do not offer any decisive advantages.</p