Background: Tissue hypoxia is a pathological condition characterized by reducing oxygen supply. Hypoxia is a hallmark of tumor environment and is commonly observed in many solid tumors. Non-invasive imaging techniques like positron emission tomography (PET) are at the forefront of detecting and monitoring tissue hypoxia changes in vivo. Results: We have developed a novel ¹⁸F-labeled radiotracer for hypoxia PET imaging based on cytotoxic agent benznidazole. Radiotracer N-(4-[¹⁸F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)acetamide ([¹⁸F]FBNA) was synthesized through acylation chemistry with readily available 4-[¹⁸F]fluorobenzyl amine. Radiotracer [¹⁸F]FBNA was obtained in good radiochemical yields (47.4 ± 5.3%) and high radiochemical purity (> 95%). The total synthesis time was 100 min, including HPLC purification and the molar activity was greater than 40 GBq/µmol. Radiotracer [¹⁸F]FBNA was stable in saline and mouse serum for 6 h. [¹⁸F]FBNA partition coefficient (logP = 1.05) was found to be more lipophilic than [¹⁸F]EF-5 (logP = 0.75), [¹⁸F]FMISO (logP = 0.4) and [¹⁸F]FAZA (logP = − 0.4). In vitro studies showed that [¹⁸F]FBNA accumulates in gastric cancer cell lines AGS and MKN45 under hypoxic conditions. Conclusions: Hence, [¹⁸F]FBNA represents a novel and easy-to-prepare PET radioligand for imaging hypoxia.

Target modules (TMs), intermediate molecules required for UniCAR T-cell therapy, are promising molecules for immunotheranostic approaches. In the current work, we developed TMs containing a monomeric or dimeric form of the antagonist bombesin peptide (BBN2) and assessed their potential for diagnostic imaging using positron emission tomography (PET) as well as immunotherapy in combination with UniCAR T-cells to target and image GRPR expression in prostate cancer. Synthesized monomeric and dimeric BBN2 TMs retained binding to GRPR in vitro. Both BBN2 TMs specifically activated and redirected UniCAR T-cells to eradicate PC3 and LNCaP cancer cells with high efficiency and in a comparable manner. UniCAR T-cells retained a non-exhausted memory phenotype favorable to their persistence and fitness. The ⁶⁸Ga-labeled BBN2 TMs showed proof-of-target towards GRPR in PC3 and LNCaP xenografts with similar uptake profiles for both BBN2 TMs in dynamic PET experiments. Clearance occurred exclusively through renal elimination. A tremendously increased in vivo metabolic stability of the BBN2 TMs was observed compared to their counterparts without E5B9. Both monomeric and dimeric BBN2 TMs represent novel and promising immunotheranostic tools for application in prostate cancer with exceptionally high in vivo metabolic stability.

The unique cell surface composition of tumor cells forms the molecular basis for many targeting and cell-based therapies. Here, we describe the development of novel peptide-based targeting agents for Programmed Death Ligand 1 (PD-L1). Molecular imaging by peptide agents, coupled with therapeutic intervention using the same modality, represents a critical advancement in cancer management. Whole-body PET imaging of PD-L1 expression offers a superior alternative to traditional immunohistochemistry, making PD-L1 radiodiagnostic imaging a highly sought-after modality. PD-L1 targeting modalities developed for clinical imaging to date can be divided into antibodies, protein domains, and small mac-rocyclic peptides with fewer than 20 amino acids. The latter modalities can address many challenges seen in antibody-based targeting vectors. All potent PD-L1 targeting peptide modalities reported to date rely extensively on non-canonical amino acids (ncAAs). Here, we report a comprehensive structure-activity relationship (SAR) analysis of a family of macrocycles discovered from an Sx2Cx8Cx2 phage-display library composed entirely of natural amino acids (x represents 19 natural amino acids excluding Cys). Using >10,000 variants in “focused” phage-display libraries, we optimized these macrocycles to achieve single-digit-nanomolar potency in protein- and cell-based assays. En route to this optimization, the activity of 216 synthetic macrocycles towards PD-L1 was measured in five distinct assays; two leads have been evaluated by imaging in tumor xenografts in mice, and the X-ray structure of one advanced lead in complex with PD-L1 has been determined at 2.78 Å resolution. This publication demonstrates the development potential of PD-L1-targeting macrocycles that do not require extensive incorporation of ncAAs and the democratization of discovery by mapping the optimization path to single-digit-nanomolar assets for targeted radiopharmaceuticals via canonical phage-display technology.