Moving Beyond Isothiocyanates: A Look at the Stability of Conjugation Links Toward Radiolysis in ⁸⁹Zr-Labeled Immunoconjugates

Zirconium-89 is the most widely used radioisotope for immunoPET because its physical half-life (78.2 h) suits the one of antibodies. Desferrioxamine B (DFO) is the standard chelator for the complexation of zirconium(IV), and its bifunctional version, containing a phenylisothiocyanate function, is the most commonly used for the conjugation of DFO to proteins. However, preliminary results have shown that the thiourea link obtained from the conjugation of isothiocyanate and lysines is sensitive to the ionizing radiation generated by the radioisotope, leading to the rupture of the link and the release of the chelator/radiometal complex. This radiolysis phenomenon could produce nonspecific signal and prevent the detection of bone metastasis, as free zirconium accumulates into the bones. The aim of this work was to study the stability of a selection of conjugation linkers in 89Zr-labeled immunoconjugates. We have synthesized several DFO-based bifunctional chelators appended with an isothiocyanate moiety, a bicyclononyne, or a squaramate ester. Two antibodies (trastuzumab and rituximab) were conjugated and radiolabeled with zirconium-89. The effect of increasing activities of zirconium-89 on the integrity of the bioconjugate bearing thiourea links was evaluated as well as the impact of the presence of a radioprotectant. The stability of the radiolabeled antibodies was studied over 7 days in PBS and human plasma. Radioconjugates’ integrity was evaluated using iTLC and size-exclusion chromatography. This study shows that the nature of the linker between the chelator and biomolecule can have a strong impact on the stability of the 89Zr-labeled conjugates, as well as on the aggregation of the conjugates

Maximum intensity projections (MIPs) PET images of ⁶⁴Cu-CC34.

<p>The images were acquired upon the injection of ⁶⁴Cu-CC34 on LNCaP tumor bearing mice at 1, 4, 24 and 48 h p.i along with blocking studies at 1h p.i.. They showed good pharmacokinetics, with clear deliniation of the tumor, gradual reduction of the kidney uptake at 24 and 48 h, respectively. The ⁶⁴Cu-labeled conjugate showed low liver uptake.</p

Internalization studies after incubation with LNCaP cells at 37°C.

<p>Approximately 75% of the total cell associated activity internalized after 3 h of incubation with the LNCaP cells Cell uptake calculated as cell surface-bound and internalized fraction. Surface bound and receptor-specific internalization expressed as percentage of the applied radioactivity. Nonspecific binding was determined in the presence of 1 μM PMPA.</p

Saturation binding study on intact LNCaP cells.

<p>Increasing concentrations of ^68/natGa-HBED-CC-PSMA, ^111/natIn-PSMA-617, ^68/natGa-CC34, ^111/natIn-CC34 and ^64/natCu-CC34 were used, ranging from 0.1 to 1,000 nM. All radiotracers exhibited high affinity for the PSMA+ LNCaP cells. ^111/natIn-CC34 and ^111/natIn-PSMA-617 are more affine to PSMA Dissociation constant (Kd) and maximum number of binding sites (Bmax) were calculated from nonlinear regression analysis using GraphPad Prism.</p

(R)-NODAGA-PSMA: A Versatile Precursor for Radiometal Labeling and Nuclear Imaging of PSMA-Positive Tumors

<div><p>Purpose</p><p>The present study aims at developing and evaluating an urea-based prostate specific membrane antigen (PSMA) inhibitor suitable for labeling with ¹¹¹In for SPECT and intraoperative applications as well as ⁶⁸Ga and ⁶⁴Cu for PET imaging.</p><p>Methods</p><p>The PSMA-based inhibitor-lysine-urea-glutamate-coupled to the spacer Phe-Phe-D-Lys(suberoyl) and functionalized with the enantiomerically pure prochelator (R)-1-(1-carboxy-3-carbotertbutoxypropyl)-4,7-carbotartbutoxymethyl)-1,4,7-triazacyclononane ((R)-NODAGA(tBu)₃), to obtain (R)-NODAGA-Phe-Phe-D-Lys(suberoyl)-Lys-urea-Glu (CC34). CC34 was labeled with ¹¹¹In, ⁶⁸Ga and ⁶⁴Cu. The radioconjugates were further evaluated in vitro and in vivo in LNCaP xenografts by biodistribution and PET studies. Biodistribution studies were also performed with ⁶⁸Ga-HBED-CC-PSMA (HBED-CC: <i>N</i>,<i>N′</i>-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-<i>N</i>,<i>N′</i>-diacetic acid) and ¹¹¹In-PSMA-617 for comparison.</p><p>Results</p><p>⁶⁸Ga-CC34, ⁶⁴Cu-CC34, and ¹¹¹In-CC34 were prepared in radiochemical purity >95%. ^68/natGa-CC34, ^64/natCu-CC34, ^111/natIn-CC34, ^68/natGa-HBED-CC-PSMA, and ^111/natIn-PSMA-617 exhibited high affinity for the LNCaP cells, with K_d values of 19.3±2.5 nM, 27.5±2.7 nM, 5.5±0.9 nM, 2.9±0.6 nM and 5.4±0.8 nM, respectively. They revealed comparable internalization profiles with approximately 75% of the total cell associated activity internalized after 3 h of incubation. ⁶⁸Ga-CC34 showed very high stability after its administration in mice. Tumor uptake of ⁶⁸Ga-CC34 (14.5±2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to ⁶⁸Ga-HBED-CC-PSMA (15.8±1.4% IA/g) (<i>P</i> = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of ⁶⁸Ga-CC34 were also comparable to ⁶⁸Ga-HBED-CC-PSMA (<i>P</i>>0.05). Tumor uptake of ¹¹¹In-CC34 (28.5±2.6% IA/g) at 1 h p.i. was lower than ¹¹¹In-PSMA-617 (52.1±6.5% IA/g) (<i>P</i> = 0.02). The acquisition of PET-images with ⁶⁴Cu-CC34 at later time points showed wash-out from the kidneys, while tumor uptake still remained relatively high. This resulted in an increased tumor-to-kidney ratio over time.</p><p>Conclusions</p><p>⁶⁸Ga-CC34 is comparable to ⁶⁸Ga-HBED-CC-PSMA in terms of tumor uptake and tumor to normal tissue ratios. ⁶⁴Cu-CC34 could enable high contrast imaging of PSMA positive tissues characterized by elevated expression of PSMA or when delayed imaging is required. ⁶⁴Cu-CC34 is currently being prepared for clinical translation.</p></div

HPLC profiles.

<p>A: formulated ⁶⁸Ga-CC34, B: mouse plasma extracted from Balb/c nude mice 15 min after injection of ⁶⁸Ga-CC34.</p

Maximum intensity projections (MIPs) PET images.

<p>(A) ⁶⁸Ga-HBED-CC-PSMA and (B) ⁶⁸Ga-CC34 upon their injection in LNCaP tumor bearing mice at 1 h and 2 h p.i along with blocking studies at 1h p.i.. The images clearly visualized the tumors. They showed high kidney uptake at early time points and prove the specificity of the radiopeptides for the PSMA receptors, as the tumor is hardly visualized after co-injection of excess of PMPA.</p

Schematic structures.

<p>(R)-NODAGA-Phe-Phe-D-Lys(suberoyl-Lys-urea-Glu) (CC34), HBED-CC-PSMA and PSMA-617.</p