Bifunctional Chelating Research Articles

P-05 - Synthesis of NOTA derivative bifunctional chelators for complexation of Re: Tc-tricarbonyl cores

P-05 - Synthesis of NOTA derivative bifunctional chelators for complexation of Re: Tc-tricarbonyl cores

O-05 - Diphosphine bifunctional chelators: [99mTc][TcO2(DP-PSMAt)2]+ tracers for PSMA imaging of prostate cancer

O-05 - Diphosphine bifunctional chelators: [99mTc][TcO2(DP-PSMAt)2]+ tracers for PSMA imaging of prostate cancer

P-74 - Curcumin-based 68Ga-radiotraces: radiolabeling strategies and bifunctional chelator approach

P-74 - Curcumin-based 68Ga-radiotraces: radiolabeling strategies and bifunctional chelator approach

Chemical and Redox Speciation of Uranyl with Three Environmentally Relevant Bifunctional Chelates: Multi-Technique Approach Combined with Theoretical Estimations.

Carbon and phosphorous are two primary elements common to the bio-geosphere and are omnipresent in both biotic and abiotic arenas. Phosphonate and carboxylate are considered as building blocks of glyphosate and humic substances and constituents of the cellular wall of bacteria and are the driving functionalities for most of the chemical interactions involving these two elements. Phosphonocarboxylates, a combination of both the functionalities in one moiety, are ideal models to dig deep into for understanding the chemical interactions of the two functional groups with metal ions. Phosphorous and carbon majorly exist as inorganic/organic phosphate and carboxylate, respectively, in the bio-geosphere. Aquatic contamination is a major concern for uranium, and the presence of complexing agents would alter the uranium concentrations in aquifers. Determination of solution thermodynamic parameters, speciation plots, redox patterns, Eh-pH diagrams, coordination structures, and molecular-level understanding by density functional theory calculations was carried out to interpret the uranyl (UO22+) interaction with three environmentally relevant phosphonocarboxylates, namely, phosphono-formic acid (PFA), phosphono-acetic acid (PAA), and phosphono-propanoic acid (PPA). UO22+ forms 1:1 complexes with the three phosphonocarboxylates in the monoprotonated form, having nearly the same stability, and the complexes [UO2(PFAH)], [UO2(PAAH)], and [UO2(PPAH)] involve chelate formation of five, six, and seven membered rings, respectively, through the participation of an oxygen each from the carboxylate and phosphonate, strengthened by an intra-molecular hydrogen bonding through the proton of the phosphonate moiety with uranyl oxygen. The complex formations are favored both enthalpically and entropically, with the latter being more contributive to the overall free energy of formation. The redox speciation showed an aqueous soluble complex formation over a wide pH range of 1-8. Electrospray ionization mass spectrometry and extended X-ray absorption fine structure established the coordination modes, which are further corroborated by density functional calculations. The knowledge gained from the present studies provide potential inputs in framing the cleanup, sequestering, microbial, and bio-remediation strategies for uranyl from aquatic environments.

Abstract A10: Radiolabelled AMD3100 for the management of malignant lymphoma: Translational study

Abstract Introduction: Malignant lymphomas are heterogeneous group of neoplasm comprising of proliferating lymphoid cells or their precursors. The two main types of lymphoma are Hodgkin Lymphoma and Non- Hodgkin Lymphoma. The current treatment modalities include chemotherapy; radiation therapy; passive immunotherapy; radio-immunotherapy and small-molecule inhibitors. Radiation therapy is the treatment of choice if disease is localized. Targeted therapy with antibody and small molecules may be more suitable for malignant lymphomas. Lymphoma cells are reported to over-express chemokine receptors that favors leukocyte infiltration, promotes tumorigenesis, angiogenesis, immune evasion, and metastasis. Human CXCL4 and CCL5 oligomerize with over 20 chemokine subfamilies to control leukocytes infiltration. CXCR4 antagonists disrupt the specific interaction of chemokines with CXCR4 and may elicit therapeutic potential. Chemokine receptor 4 (CXCR4) has been explored with radiolabelled CXCR4 antagonist (AMD3100) for imaging and future targeted radiation therapy for malignant lymphoma. Methods: Standardization of conjugation of AMD3100 with bifunctional chelating agents (DTPA, NOTA etc.). Optimization include radiolabelling of conjugated AMD3100 with 68Ga and 177Lu for imaging and therapeutic purpose respectively. The radionuclide and radiochemical purity of the product was determined. CXCR4 binding efficacy and toxicity studies were performed on CXCR4 expressing cancer cell-lines. The quality control of radiolabelled AMD3100 included, radiochemical purity, sterility, pyrogenicity, human serum and PBS stability. The biodistribution studies were performed in normal rats. After obtaining clearance from institutional ethics committee,68Ga-AMD3100-DTPA PET/CT was performed in lymphoma patients (n=2). The uptake was compared with 18FDG uptake for proof of concept. Results: DTPA conjugation of AMD3100 (1087 Da) and NOTA conjugation (1014 Da) was confirmed with MALDI-TOF. The radionuclide and radiochemical purity of 68Ga and 177Lu AMD3100 was >99%. Radio-ligand binding assay confirmed high specificity (Kd = 57.16 nM) of 177Lu-AMD3100-DTPA towards CXCR4 expressing cancer cells. Furthermore, the log absolute IC50 concentration of 177Lu-AMD3100-DTPA calculated for cytotoxicity assessment, via MTT assay, was 2.628 nM. Immunocytochemistry depicted positive nuclear staining of CXCR4 receptors in cancer cells. The synthesized radiopharmaceuticals were sterile and pyrogen-free. In-vivo physiological biodistribution of 68Ga-AMD3100-DTPA was found in liver, lung and spleen. The radiotracer showed faster renal clearance and low blood pool activity. In addition to physiological uptake, 68Ga-AMD3100-DTPA PET/CT showed similar lesions that were seen in 18FDG PET/CT of lymphoma patients. Conclusion: 68Ga/177Lu-AMD3100-DTPA demonstrated high target specificity towards CXCR4 expressing lymphoma cells. Cytotoxicity and patient study indicated its potential as theranostic agent. It can be used for the selection of patients, targeted therapy and response evaluation. Citation Format: Tamanna Lakhanpal, Jaya Shukla, Rajender Kumar, Harmanpreet Singh, Pankaj Malhotra, Alka Khadwal, Gaurav Prakash, Amanjit Bal, Yogesh Rathore, BR Mittal. Radiolabelled AMD3100 for the management of malignant lymphoma: Translational study [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A10.

Development of sensitive and portable immunosensors based on signal amplification probes for monitoring the mercury(II) ions

Mercury ion (Hg2+) as a major environmental pollutant threatens human health even at very low concentrations, so it is essential to monitor mercury residues in food. In this study, Hg2+ was conjugated with protein carrier using 1-(4-Isothiocyanobenzyl) ethylenediamine N, N, N′, N′-tetraacetic acid (ITCBE) as a bifunctional chelator. 7A1 monoclonal antibody (mAb) against Hg2+-ITCBE with high affinity (7.3 × 109 L/moL) and good specificity was obtained by cell fusion technology and performed to establish immunosensors. Immunochromatographic test strip using colloidal gold nanoparticles (AuNP with an average diameter of 18 nm) as signal reporter showed low sensitivity. Signal amplification probes including larger multi-branched gold nanoflowers (AuNF) and latex microspheres (LM) were employed to enhance the sensitivity of immunosensors. The visible limit of detection (vLOD) of the AuNF- and LM-based strip were determined to be 50 ng/mL and 25 ng/mL respectively, showing more sensitive than that of AuNP-based strip (200 ng/mL). Quantitative analysis showed that AuNF-based strip exhibited lower quantitative limit of detection (qLOD) (0.44 ng/mL) which was 20-fold lower than that of AuNP-based strip (8.92 ng/mL) for determination of Hg2+, and LM-based strip (0.49 ng/mL) was 18 times as sensitive as AuNP-based strip. In summary, the developed immunosensors using AuNF and LM as signal amplification probes exhibited excellent sensitivity and provided portable, on-site detection for Hg2+.

Preclinical Assessment of [68Ga]Ga-Cell Death Indicator (CDI): A Novel hsp90 Ligand for Positron Emission Tomography of Cell Death.

4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO) when conjugated with a bifunctional chelator 2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4- oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODAGA) (hereafter referred to as Cell Death Indicator [CDI]), enters dead and dying cells and binds to 90kDa heat shock proteins (hsp90). This study assesses stability, biodistribution, imaging, and radiation dosimetry of [68Ga]- Ga-CDI for positron emission tomography (PET). Preparation of [68Ga]Ga-CDI was performed as previously described. Product stability and stability in plasma were assessed using high-performance liquid chromatography. Biodistribution and imaging were conducted in ten healthy male Lewis rats at 1 and 2 h following intravenous [68Ga]Ga-CDI injection. Human radiation dosimetry was estimated by extrapolation for a standard reference man and calculated with OLINDA/EXM 1.1. Radiochemical purity of [68Ga]Ga-CDI averaged 93.8% in the product and 86.7% in plasma at 4 h post-synthesis. The highest concentration of [68Ga]Ga-CDI is observed in the kidneys; [68Ga]Ga-CDI is excreted in the urine, and mean retained activity was 32.4% and 21.4% at 1 and 2 h post-injection. Lower concentrations of [68Ga]Ga-CDI were present in the small bowel and liver. PET CT was concordant and additionally demonstrated focal growth plate uptake. The effective dose for [68Ga]Ga-CDI is 2.16E-02 mSv/MBq, and the urinary bladder wall received the highest dose (1.65E-02 mSv/Mbq). [68Ga] Ga-CDI is stable and has favourable biodistribution, imaging, and radiation dosimetry for imaging of dead and dying cells. Human studies are underway.

Synthesis and evaluation of a para-carboxylated benzyl-DOTA for labeling peptides and polypeptides

IntroductionRadiolabeled peptides and low-molecular-weight (LMW) polypeptides show high and persistent radioactivity levels in the kidney. To develop a DOTA-based bifunctional chelating agent that provides a radiometabolite with a rapid elimination rate from the kidney, a para-carboxyl Bn-DOTA (p-COOH-Bn-DOTA) was designed, synthesized, and evaluated. MethodsA precursor compound, p-COOH-Bn-DOTA(tBu)4, was synthesized in 9 steps using N-Boc-p-iodo-L-phenylalanine as the starting material. A synthetic somatostatin analog (TOC) was used as a representative peptide metabolized in the renal lysosomes. p-COOH-Bn-DOTA-conjugated TOC (DOTA-Bn-TOC) was synthesized by the conventional solid-phase peptide synthesis using p-COOH-Bn-DOTA(tBu)4. DOTA-tris(tBu ester) was also conjugated with TOC to prepare DOTATOC. 111In-labeling of the peptides was conducted under similar conditions. The radiochemical conversions, stability against apo-transferrin (apoTf), and in vivo behaviors were compared. Results[111In]In-DOTA-Bn-TOC was obtained with higher radiochemical conversions than [111In]In-DOTATOC. Both 111In-labeled TOC derivatives remained stable against apoTf. In biodistribution studies, [111In]In-DOTA-Bn-TOC exhibited higher initial uptake in the kidney, followed by a faster elimination rate of radioactivity into the urine than [111In]In-DOTATOC. The metabolic studies showed that the shorter residence time of the radiometabolite from [111In]In-DOTA-Bn-TOC was responsible for the renal radioactivity decline. Conclusionp-COOH-Bn-DOTA provides stable 111In-labeled peptides in high yields at low peptide concentrations. p-COOH-Bn-DOTA also provides a radiometabolite with a short residence time in the kidney. Such characteristics would render p-COOH-Bn-DOTA useful to the future application to radiolabeled LMW polypeptides with low renal radioactivity levels.

Preclinical Evaluation of hnRNPA2B1 Antibody in Human Triple-Negative Breast Cancer MDA-MB-231 Cells via PET Imaging.

Triple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Because TNBC lacks the expression of commonly targeted receptors, it is challenging to develop a new imaging agent for this cancer subtype. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA–protein complexes that have been linked to tumor development and progression. Considering the high expression of hnRNPA2B1, an hnRNP subtype, in TNBC MDA-MB-231 cells, this study aimed to develop a novel hnRNPA2B1 antibody-based nuclear imaging agent. The hnRNPA2B1-specific antibody was radiolabeled with 64Cu and evaluated in vitro and in vivo. The trans-cyclooctene (TCO) was functionalized on the antibody to obtain hnRNP-PEG4-TCO and reactive tetrazine (Tz) on the ultrastable bifunctional chelator PCB-TE2A-alkyne to yield PCB-TE2A-Tz for the inverse electron demand Diels–Alder reaction. The 64Cu-radiolabeled antibody was administered and imaged at 1–18 h time points for conventional imaging. Alternatively, the unlabeled antibody conjugate was administered, and 48 h later radiolabeled 64Cu-PCB-TE2A-Tz was administered to the same mice for the pretargeting strategy and imaged at the same time intervals for direct comparison. The tumor was successfully visualized in both strategies, and comparatively, pretargeting showed superior results. The 64Cu-PCB-TE2A-Tz was successfully clicked at the tumor site with hnRNP-PEG4-TCO and the non-clicked were concurrently eliminated. This led to increase the tumor uptake with extremely high tumor-to-background ratio manifested by positron emission tomography (PET) imaging and biodistribution studies.

Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates.

The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimal in vitro and in vivo behavior. In response to this, several site-selective approaches to bioconjugation have been developed, yet each has intrinsic drawbacks, such as the need for expensive reagents or the complexity of incorporating unnatural amino acids into IgGs. Herein, we describe the use of a simple and facile approach to lysine-directed site-selective bioconjugation for the generation of radioimmunoconjugates. This strategy relies upon on the selective modification of single lysine residues within each light chain of the monoclonal antibody (mAb) with a branched azide-bearing perfluorophenyl ester (PFP-bisN3) followed by the ligation of dibenzocyclooctyne (DBCO)-bearing payloads to these bioorthogonal handles via the strain-promoted azide-alkyne cycloaddition. This methodology was used to create [89Zr]Zr-SSKDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (89Zr). [89Zr]Zr-SSKDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([89Zr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([89Zr]Zr-malDFO-pertuzumab). The bioconjugation strategy was assessed using ESI mass spectrometry, SDS-PAGE, and autoradiography. All three immunoconjugates demonstrated comparable binding to HER2 via flow cytometry and surface plasmon resonance (SPR), and 89Zr-labeled variants of each were synthesized in >99% radiochemical yield and molar activities of up to ∼55.5 GBq/μmol (10 mCi/mg). Subsequently, the in vivo behavior of this trio of 89Zr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab produced positron emission tomography (PET) images with high tumoral uptake and high tumor-to-healthy organ activity concentration ratios. A terminal biodistribution study complemented the PET results, revealing tumoral activity concentrations of 126.9 ± 50.3%ID/g, 86.9 ± 53.2%ID/g, and 92.5 ± 27.2%ID/g at 144 h post-injection for [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab, respectively. Taken together, the data clearly illustrate that this highly modular and facile approach to site-selective bioconjugation produces radioimmunoconjugates that are better-defined and more homogeneous than stochastically modified constructs and also exhibit excellent in vitro and in vivo performance. Furthermore, we contend that this lysine-directed strategy holds several key advantages over extant approaches to site-selective bioconjugation, especially in the context of production for the clinic.

On the dissociation pathways of copper complexes relevant as PET imaging agents

Several bifunctional chelators have been synthesized in the last years for the development of new 64Cu-based PET agents for in vivo imaging. When designing a metal-based PET probe, it is important to achieve high stability and kinetic inertness once the radioisotope is coordinated. Different competitive assays are commonly used to evaluate the possible dissociation mechanisms that may induce Cu(II) release in the body. Among them, acid-assisted dissociation tests or transchelation challenges employing EDTA or SOD are frequently used to evaluate both solution thermodynamics and the kinetic behavior of potential metal-based systems. Despite of this, the Cu(II)/Cu(I) bioreduction pathway that could be promoted by the presence of bioreductants still remains little explored. To fill this gap we present here a detailed spectroscopic study of the kinetic behavior of different macrocyclic Cu(II) complexes. The complexes investigated include the cross-bridge cyclam derivative [Cu(CB-TE1A)]+, whose structure was determined using single-crystal X-ray diffraction. The acid-assisted dissociation mechanism was investigated using HClO4 and HCl to analyse the effect of the counterion on the rate constants. The complexes were selected so that the effects of complex charge and coordination polyhedron could be assessed. Cyclic voltammetry experiments were conducted to investigate whether the reduction to Cu(I) falls within the window of common bioreducing agents. The most striking behavior concerns the [Cu(NO2Th)]2+ complex, a 1,4,7-triazacyclononane derivative containing two methylthiazolyl pendant arms. This complex is extremely inert with respect to dissociation following the acid-catalyzed mechanism, but dissociates rather quickly in the presence of a bioreductant like ascorbic acid.

Trastuzumab-conjugated oxine-based ligand for [89Zr]Zr4+ immunoPET

A new, bifunctional chelating ligand for immuno-Positron Emission Tomography (PET) was designed, synthesized, and conjugated to Trastuzumab for a proof-of-concept study with 89Zr. H4neunox was synthesized from the tris(2-aminoethyl)amine backbone, decorated with 8-hydroxyquinoline moieties, and utilizes a primary amine for functionalization. A maleimide moiety extends the chelator to create H4neunox-mal for antibody conjugation via maleimide-thiol click chemistry. Preliminary 89Zr radiolabeling of H4neunox indicated quantitative radiolabeling at 1 × 10−5 M, but improved inertness towards human serum (96% intact at 7 d) and Fe3+ (92% intact at 24 h) compared to the previously synthesized H5decaox. The chelator was successfully conjugated to the monoclonal antibody, Trastuzumab, and used in preliminary radiolabeling reactions (37 °C, 2 h) with 89Zr. Radiochemical assessments of the new H4neunox-Trastuzumab conjugate include 89Zr radiolabeling, spin filter purification, cell-binding immunoreactivity, and in vivo PET imaging and biodistribution in SKOV-3 tumour bearing nude mice, performed in comparison with the desferrioxamine B analog, DFO-Trastuzumab. The [89Zr]Zr(neunox-Trastuzumab) showed lowered inertness towards serum (76% intact at 24 h) as well as demetallation in vivo through bone uptake (21% ID/g) in PET imaging and biodistribution studies when compared to [89Zr]Zr(DFO-Trastuzumab). Although the combination of the chelator and antibody had detrimental effects on their intended purposes, nonetheless, the primary amine platform of H4neunox developed here provides an oxine-based bifunctional ligand for further derivatizations with other targeting vectors.

Toward Bifunctional Chelators for Thallium-201 for Use in Nuclear Medicine.

Auger electron therapy exploits the cytotoxicity of low-energy electrons emitted during radioactive decay that travel very short distances (typically <1 μm). 201Tl, with a half-life of 73 h, emits ∼37 Auger and other secondary electrons per decay and can be tracked in vivo as its gamma emissions enable SPECT imaging. Despite the useful nuclear properties of 201Tl, satisfactory bifunctional chelators to incorporate it into bioconjugates for molecular targeting have not been developed. H4pypa, H5decapa, H4neunpa-NH2, and H4noneunpa are multidentate N- and O-donor chelators that have previously been shown to have high affinity for 111In, 177Lu, and 89Zr. Herein, we report the synthesis and serum stability of [nat/201Tl]Tl3+ complexes with H4pypa, H5decapa, H4neunpa-NH2, and H4noneunpa. All ligands quickly and efficiently formed complexes with [201Tl]Tl3+ that gave simple single-peak radiochromatograms and showed greatly improved serum stability compared to DOTA and DTPA. [natTl]Tl-pypa was further characterized using nuclear magnetic resonance spectroscopy (NMR), mass spectroscopy (MS), and X-ray crystallography, showing evidence of the proton-dependent presence of a nine-coordinate complex and an eight-coordinate complex with a pendant carboxylic acid group. A prostate-specific membrane antigen (PSMA)-targeting bioconjugate of H4pypa was synthesized and radiolabeled. The uptake of [201Tl]Tl-pypa-PSMA in DU145 PSMA-positive and PSMA-negative prostate cancer cells was evaluated in vitro and showed evidence of bioreductive release of 201Tl and cellular uptake characteristic of unchelated [201Tl]TlCl. SPECT/CT imaging was used to probe the in vivo biodistribution and stability of [201Tl]Tl-pypa-PSMA. In healthy animals, [201Tl]Tl-pypa-PSMA did not show the myocardial uptake that is characteristic of unchelated 201Tl. In mice bearing DU145 PSMA-positive and PSMA-negative prostate cancer xenografts, the uptake of [201Tl]Tl-pypa-PSMA in DU145 PSMA-positive tumors was higher than that in DU145 PSMA-negative tumors but insufficient for useful tumor targeting. We conclude that H4pypa and related ligands represent an advance compared to conventional radiometal chelators such as DOTA and DTPA for Tl3+ chelation but do not resist dissociation for long periods in the biological environment due to vulnerability to reduction of Tl3+ and subsequent release of Tl+. However, this is the first report describing the incorporation of [201Tl]Tl3+ into a chelator–peptide bioconjugate and represents a significant advance in the field of 201Tl-based radiopharmaceuticals. The design of the next generation of chelators must include features to mitigate this susceptibility to bioreduction, which does not arise for other trivalent heavy radiometals.

Development of [18F]AmBF3 Tetrazine for Radiolabeling of Peptides: Preclinical Evaluation and PET Imaging of [18F]AmBF3-PEG7-Tyr3-Octreotide in an AR42J Pancreatic Carcinoma Model.

Radiolabeled peptides have emerged as highly specific agents for targeting receptors expressed in tumors for therapeutic and diagnostic purposes. Peptides developed for positron emission tomography (PET) are typically radiolabeled using prosthetic groups or bifunctional chelators for fast “kit-like” incorporation of the radionuclide into the structure. A novel [18F]alkylammoniomethyltrifluoroborate ([18F]AmBF3) tetrazine (Tz), [18F]AmBF3-Tz, was developed for the [18F]fluorination of trans-cyclooctene (TCO)-modified biomolecules using Tyr3-octreotides (TOCs) as model peptides. [18F]AmBF3-Tz (Am = 15.4 ± 9.2 GBq/μmol, n = 14) was evaluated in healthy mice by ex vivo biodistribution and PET/computed tomography (CT), where the radiolabel in the prosthetic group was found stable in vivo, indicated by the low bone uptake in tibia (0.4 ± 0.1% ID/g, t = 270 min). TCO-TOCs tailored with polyethylene glycol (PEG) linkers were radiolabeled with [18F]AmBF3-Tz, forming two new tracers, [18F]AmBF3-PEG4-TOC (Am = 2.8 ± 1.8 GBq/μmol, n = 3) and [18F]AmBF3-PEG7-TOC (Am of 6.0 ± 3.4 GBq/μmol, n = 13), which were evaluated by cell uptake studies and ex vivo biodistribution in subcutaneous AR42J rat pancreatic carcinoma tumor-bearing nude mice. The tracer demonstrating superior behavior ex vivo, the [18F]AmBF3-PEG7-TOC, was further evaluated with PET/CT, where the tracer provided clear tumor visualization (SUVbaseline = 1.01 ± 0.07, vs SUVblocked = 0.76 ± 0.04) at 25 min post injection. The novel AmBF3-Tz demonstrated that it offers potential as a prosthetic group for rapid radiolabeling of biomolecules in mild conditions using bioorthogonal chemistry.

Abstract 1401: Lead optimization of radiopharmaceuticals for molecular radiotherapy and preclinical evaluation

Abstract Molecular Radiotherapy (MRT) targeting SSTR2 or PSMA have proven to be highly efficient for the treatment of neuroendocrine or metastatic prostate cancer, respectively. Beyond the leading radiopharmaceutical molecules 177Lu-DOTATATE or 177Lu-PSMA-617, a variety of vectors (small molecules, peptides, panel of biologics) have been developed on the same targets in order to improve the biodistribution within the tumor, the blood clearance, the route of elimination or the dosimetry. The labeling of the targeting ligand, whatever its nature, is a crucial step as it may affect significantly the properties of the theranostic conjugate, i.e. its binding affinity, PK and biodistribution. The addition of linkers, such as albumin binding domain or PEG, and choice of chelating agents have a major impact on the chemical and biological properties of the vectors. Random or site-specific bioconjugation, click chemistry, also have to be considered in the early stage as the choice of the selected technology will modify your development plan and manufacturing. New ligands and biological platforms are now being developed based on this historical knowledge, improved Target Product Profiles are built to conduct optimal lead optimization of MRT. Herein, we will present our lead optimization and preclinical evaluation process to select efficiently good radiolabeled molecules and list the key parameters to be checked. To date, it remains hard to predict the behavior of the modified bioconjugated molecules, and versatile synthesis strategies are needed to screen various combinations of radiometal complexes, linker and conjugation function, in order to converge rapidly to the optimized bioconjugate. As an example, we will present a study case where the conjugation of various bifunctional chelating agents on a small NTS1 receptor antagonist resulted in drastically different in vivo behavior of the resulting 68Ga-labeled compounds. Once optimal in vivo tumor uptake has been achieved, preclinical evaluation requires the selection of appropriate and relevant models, driven by target expression, radioresistance, and potentially tumor immune infiltrate for combination studies with immunotherapies. The therapeutic evaluation should take into consideration the dose and specific activity, the tolerance of a model related to ionizing radiations and the scheduling of treatments (cumulated dose, fractionation). We will present our recent results which highlight the importance to optimize these parameters to improve the efficacy of MRT. Citation Format: Emma Renard, Olivier Raguin, Victor Goncalves, Celine Mothes, Mathieu Moreau, Claire Bernhard, Peggy Provent, Frederic Boschetti, Franck Denat, Fabrice Viviani, Cyril Berthet. Lead optimization of radiopharmaceuticals for molecular radiotherapy and preclinical evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1401.

Abstract 1234: PD-L1 PET: A potential biomarker of checkpoint inhibitor sensitivity in renal cancer

Abstract Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. Program death-ligand 1 (PD-L1) and its corresponding receptor (PD-1) are the target of most FDA-approved ICIs. Perhaps unsurprisingly, PD-L1 levels are predictive of response to targeting drugs in lung and other cancers. However, PD-L1 is not predictive in other tumor types including renal cell carcinoma (RCC). PD-L1 is typically measured on tumor biopsies or archival tissues and we hypothesized that the lack of PD-L1 predictive power is due to tumor heterogeneity and tumor evolution. To test this notion, we embarked upon the generation of a PD-L1 probe that would enable real time assessment of PD-L1 across sites of disease using molecular imaging. We leveraged atezolizumab, a highly specific PD-L1 therapeutic antibody with a mutant Fc. Atezolizumab (ATZ) was labeled with 89Zr after conjugation with a bifunctional chelator and evaluated by positron emission tomography (PET). In extensive preclinical studies using multiple patient-derived xenografts (PDXs) with variable PD-L1 levels, we show that 89Zr-ATZ PET is able to distinguish RCC with variable PD-L1 expression. These data set the foundation for an investigator new drug (IND) approval from the FDA and an ongoing clinical trial in RCC patients (NCT04006522). Consistent with our hypothesis, 89Zr-ATZ PET shows substantial PD-L1 heterogeneity not only across but also within patients at different sites of disease. Furthermore, preliminary data suggest that PD-L1 levels may be predictive of response to ICI. By assessing PD-L1 expression in real time, 89Zr-ATZ PET may enable: (1) the identification of patients most likely to respond to PD-L1/PD-1 targeted therapies; (2) tailored management of the disease across different sites; (3) the evaluation of interventions that modulate PD-L1 levels; (4) insights into toxicities; and (5) probing resistance. Citation Format: Aditi Mulgaonkar, Layton Woolford, Roy Elias, Kien Nham, Bing Guan, Guiyang Hao, Christina Stevens, Vanina Toffessi Tcheuyap, Sydney Haldeman, Jeffrey Miyata, Deyssy Carrillo, Qian (Janie) Qin, Nirmish Singla, Isaac Bowman, Jeffrey Cadeddu, Vitaly Margulis, Alana Christie, Payal Kapur, Ivan Pedrosa, Marianna Dakanali, Orhan Oz, Xiankai Sun, James Brugarolas. PD-L1 PET: A potential biomarker of checkpoint inhibitor sensitivity in renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1234.

A Narrative Review of 99mTc-Aprotinin in the Diagnosis of Cardiac Amyloidosis and a New Life for an Unfairly Abandoned Drug

Several studies investigated the use of 99mTc-labelled Aprotinin as an amyloid seeker some years ago. In vitro tests showed high binding affinity for several types of amyloid fibrils accompanied by an excellent specificity. Initial human studies demonstrated good accuracy in detecting cardiac involvement. Scintigraphy results were confirmed in a group of 28 endomyocardial biopsies. Unfortunately, clinical studies were halted because of a temporary suspension of the vector protein (Trasylol) and public health concerns over prion contamination of the bovine origin compound. To obviate these limitations, efforts have been made to label a recombinant Aprotinin with 99mTc, which exhibits the same affinity for h-insulin fibrils. With the aim of developing a PET tracer, the same recombinant protein was labeled with Gallium. The introduction of a bifunctional chelator did not affect fibril affinity. Finally, a synthetic peptidic fragment, the cyclic 30-51 SS, was synthetized. After direct technetium labeling, an impressive increase in affinity was demonstrated. This peptide appears to be a potential candidate for Gallium labeling through a bifunctional chelator for PET imaging.

H2BZmacropa-NCS: A Bifunctional Chelator for Actinium-225 Targeted Alpha Therapy.

Actinium-225 (225Ac) is one of the most promising radionuclides for targeted alpha therapy (TAT). With a half-life of 9.92 days and a decay chain that emits four high-energy α particles, 225Ac is well-suited for TAT when conjugated to macromolecular targeting vectors that exhibit extended in vivo circulation times. The implementation of 225Ac in these targeted constructs, however, requires a suitable chelator that can bind and retain this radionuclide in vivo. Previous work has demonstrated the suitability of a diaza-18-crown-6 macrocyclic chelator H2macropa for this application. Building upon these prior efforts, in this study, two rigid variants of H2macropa, which contain either one (H2BZmacropa) or two (H2BZ2macropa) benzene rings within the macrocyclic core, were synthesized and investigated for their potential use for 225Ac TAT. The coordination chemistry of these ligands with La3+, used as a nonradioactive model for Ac3+, was carried out. Both NMR spectroscopic and X-ray crystallographic studies of the La3+ complexes of these ligands revealed similar structural features to those found for the related complex of H2macropa. Thermodynamic stability constants of the La3+ complexes, however, were found to be 1 and 2 orders of magnitude lower than those of H2macropa for H2BZmacropa and H2BZ2macropa, respectively. The decrease in thermodynamic stability was rationalized via the use of density functional theory calculations. 225Ac radiolabeling and serum stability studies with H2BZmacropa showed that this chelator compares favorably with H2macropa. Based on these promising results, a bifunctional version of this chelator, H2BZmacropa-NCS, was synthesized and conjugated to the antibody codrituzumab (GC33), which targets the liver cancer biomarker glypican-3 (GPC3). The resulting GC33-BZmacropa conjugate and an analogous GC33-macropa conjugate were evaluated for their 225Ac radiolabeling efficiencies, antigen-binding affinities, and in vivo biodistribution in HepG2 liver cancer tumor-bearing mice. Although both conjugates were comparably effective in their radiolabeling efficiencies, [225Ac]Ac-GC33-BZmacropa showed slightly poorer serum stability and biodistribution than [225Ac]Ac-GC33-macropa. Together, these results establish H2BZmacropa-NCS as a new bifunctional chelator for the preparation of 225Ac radiopharmaceuticals.

68Ga-Labeled Benzothiazole Derivatives for Imaging Aβ Plaques in Cerebral Amyloid Angiopathy.

Timely diagnostic imaging plays a crucial role in managing cerebral amyloid angiopathy (CAA)—the condition in which amyloid β is deposited on blood vessels. To selectively map these amyloid plaques, we have designed amyloid-targeting ligands that can effectively complex with 68Ga3+ while maintaining good affinity for amyloid β. In this study, we introduced novel 1,4,7-triazacyclononane-based bifunctional chelators (BFCs) that incorporate a benzothiazole moiety as the Aβ-binding fragment and form charged and neutral species with 68Ga3+. In vitro autoradiography using 5xFAD and WT mouse brain sections (11-month-old) suggested strong and specific binding of the 68Ga complexes to amyloid β. Biodistribution studies in CD-1 mice revealed a low brain uptake of 0.10–0.33% ID/g, thus suggesting 68Ga-labeled novel BFCs as promising candidates for detecting CAA.

Bioconjugated chelates based on (methylpyridinyl)tacn: synthesis, 64Cu labeling and in vitro evaluation for prostate cancer targeting.

Three new bifunctional copper chelators based on the 1,4,7-triazacyclononane (tacn) platform have been synthesized and conjugated to peptides. The first one is constituted of the tacn with two methylpyridinyl and one methylthiazolyl carboxylic acid pendant arms, while, in the second and third ones, the macrocycle is functionalized by three methylpyridinyl groups, with an additional hexynoic acid chain on a carbon of one or two pyridine rings. These three bifunctional chelators have been conjugated to the antagonist DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 peptide for targeting the gastrin-releasing peptide receptor, which is overexpressed in prostate cancer. The resulting monomeric bioconjugates have shown their efficiency to be radiolabeled with β+ emitter 64Cu, and the hydrophilicity and PC-3 cell internalization properties of these radiolabeled conjugates have been studied. PC-3 cell binding affinity of mono- and dimeric metal-free and natCu metallated conjugates have been evaluated by IC50 measurements. The results demonstrate the potential of these methylpyridinyl tacn derivatives for radiopharmaceutical applications.