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While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

Despite a rise in clinical use of radiopharmaceutical therapies, the biological effects of radionuclides and their relationship with absorbed radiation dose are poorly understood. Here, we set out to define this relationship for Auger electron emitters [99mTc]TcO 4 − and [123I]I− and β−-particle emitter [188Re]ReO 4 −. Studies were carried out using genetically modified cells that permitted direct radionuclide comparisons. Triple-negative MDA-MB-231 breast cancer cells expressing the human sodium iodide symporter (hNIS) and green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used. In vitro radiotoxicity of [99mTc]TcO 4 −, [123I]I−, and [188Re]ReO 4 − was determined using clonogenic assays. Radionuclide uptake, efflux, and subcellular location were used to calculate nuclear absorbed doses using the Medical Internal Radiation Dose (MIRD) formalism. In vivo studies were performed using female NSG mice bearing orthotopic MDA-MB-231.hNIS-GFP tumors and compared with X-ray–treated (12.6-15 Gy) and untreated cohorts. Absorbed dose per unit activity in tumors and sodium iodide symporter–expressing organs was extrapolated to reference human adult models using OLINDA/EXM. [99mTc]TcO 4 − and [123I]I− reduced the survival fraction only in hNIS-expressing cells, whereas [188Re]ReO 4 − reduced survival fraction in hNIS-expressing and parental cells. [123I]I− required 2.4- and 1.5-fold lower decays/cell to achieve 37% survival compared with [99mTc]TcO 4 − and [188Re]ReO 4 −, respectively, after 72 hours of incubation. Additionally, [99mTc]TcO 4 −, [123I]I−, and [188Re]ReO 4 − had superior cell killing effectiveness in vitro compared with X-rays. In vivo, X-ray led to a greater median survival compared with [188Re]ReO 4 − and [123I]I− (54 days vs 45 and 43 days, respectively). Unlike the X-ray cohort, no metastases were visualized in the radionuclide-treated cohorts. Extrapolated human absorbed doses of [188Re]ReO 4 − to a 1 g tumor were 13.8- and 11.2-fold greater than for [123I]I− in female and male models, respectively. This work reports reference dose-effect data using cell and tumor models for [99mTc]TcO 4 −, [123I]I−, and [188Re]ReO 4 − for the first time. We further demonstrate the tumor-controlling effects of [123I]I− and [188Re]ReO 4 − in comparison with external beam radiation therapy. [ABSTRACT FROM AUTHOR]

The π-clamp-mediated conjugation method, which enables site-specific modification of cysteine residues, is a promising strategy for developing well-defined radiolabelled biomolecules for positron emission tomography (PET) imaging. We have applied this method to site-specifically attach the macrocyclic chelators "NODA" and "NODAGA" to the somatostatin receptor 2-targeted peptide, octreotate. The resulting novel NODA-octreotate and NODAGA-octreotate compounds can be radiolabelled with either [ 18 F]AlF - or [ 68 Ga]Ga 3+ respectively. In vivo PET imaging shows that the [ 68 Ga]Ga 3+ -labelled derivative exhibits high stability and favourable pharmacokinetic properties.

Natural killer (NK) cells can kill cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC): a tumor-associated IgG antibody binds to the Fcγ receptor CD16 on NK cells via the antibody Fc region and activates the cytotoxic functions of the NK cell. Here, we used PET imaging to assess NK cell migration to human epidermal growth factor receptor 2 (HER2)-positive HCC1954 breast tumors, examining the influence of HER2-targeted trastuzumab antibody treatment on NK cell tumor accumulation. Methods: Human NK cells from healthy donors were expanded ex vivo and labeled with [ 89 Zr]Zr-oxine. In vitro experiments compared the phenotypic markers, viability, proliferation, migration, degranulation, and ADCC behaviors of both labeled ( 89 Zr-NK) and unlabeled NK cells. Female mice bearing orthotopic human breast HCC1954 tumors were administered 89 Zr-NK cells alongside trastuzumab treatment or a sham treatment and then scanned using PET/CT imaging over 7 d. Flow cytometry and γ-counting were used to analyze the presence of 89 Zr-NK cells in liver and spleen tissues. Results: 89 Zr cell radiolabeling yields measured 42.2% ± 8.0%. At an average specific activity of 16.7 ± 4.7 kBq/10 6 cells, 89 Zr-NK cells retained phenotypic and functional characteristics including CD56 and CD16 expression, viability, migration, degranulation, and ADCC capabilities. In vivo PET/CT studies indicated predominant accumulation of 89 Zr-NK cells in the liver and spleen. Ex vivo analyses of liver and spleen tissues indicated that the administered human 89 Zr-NK cells retained their radioactivity in vivo and that 89 Zr did not transfer to cells of murine soft tissues, thus validating this 89 Zr PET method for NK cell tracking. Notably, 89 Zr-NK cells migrated to HER2-positive tumors, both with and without trastuzumab treatment. Trastuzumab treatment was associated with an increased 89 Zr-NK cell signal at days 1 and 3 after injection. Conclusion: In vitro, 89 Zr-NK cells maintained key cellular and cytotoxic functions. In vivo, 89 Zr-NK cells trafficked to HER2-postive tumors, with trastuzumab treatment correlating with enhanced 89 Zr-NK infiltration. This study demonstrates the feasibility of using PET to image 89 Zr-NK cell infiltration into solid tumors., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Benchtop 99 Mo/ 99m Tc and 188 W/ 188 Re generators enable economical production of molecular theranostic 99m Tc and 188 Re radiopharmaceuticals, provided that simple, kit-based chemistry exists to radiolabel targeting vectors with these radionuclides. We have previously described a diphosphine platform that efficiently incorporates 99m Tc into receptor-targeted peptides. Here, we report its application to label a prostate-specific membrane antigen (PSMA)-targeted peptide with 99m Tc and 188 Re for diagnostic imaging and systemic radiotherapy of prostate cancer. Methods: Two diphosphine-dipeptide bioconjugates, DP1-PSMAt and DP2-PSMAt, were formulated into kits for radiolabeling with 99m Tc and 188 Re. The resulting radiotracers were studied in vitro, in prostate cancer cells, and in vivo in mouse xenograft models, to assess similarity of uptake and biodistribution for each 99m Tc/ 188 Re pair of agents. Results: Both DP1-PSMAt and DP2-PSMAt could be efficiently radiolabeled with 99m Tc and 188 Re using kit-based methods to furnish the isostructural compounds M-DP1-PSMAt and M-DP2-PSMAt (M = [ 99m Tc]Tc, [ 188 Re]Re). All 99m Tc/ 188 Re radiotracers demonstrated specific uptake in PSMA-expressing prostate cancer cells, with negligible uptake in prostate cancer cells that did not express PSMA or in which PSMA uptake was blocked. M-DP1-PSMAt and M-DP2-PSMAt also exhibited high tumor uptake (18-30 percentage injected dose per gram at 2 h after injection), low retention in nontarget organs, fast blood clearance, and excretion predominantly via a renal pathway. Importantly, each pair of 99m Tc/ 188 Re radiotracers showed near-identical biologic behavior in these experiments. Conclusion: We have prepared and developed novel pairs of isostructural PSMA-targeting 99m Tc/ 188 Re theranostic agents. These generator-based theranostic agents have potential to provide access to the benefits of PSMA-targeted diagnostic imaging and systemic radiotherapy in health care settings that do not routinely have access to either reactor-produced 177 Lu radiopharmaceuticals or PET/CT infrastructure., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Bioconjugates of antibodies and their derivatives radiolabeled with β + -emitting radionuclides can be utilized for diagnostic PET imaging. Site-specific attachment of radioactive cargo to antibody delivery vectors provides homogeneous, well-defined immunoconjugates. Recent studies have demonstrated the utility of oxaziridine chemistry for site-specific labeling of methionine residues. Herein, we applied this approach to site-specifically radiolabel trastuzumab-derived Fab immunoconjugates with 68 Ga, which can be used for in vivo PET imaging of HER2-positive breast cancer tumors. Initially, a reactive azide was introduced to a single solvent-accessible methionine residue in both the wild-type Fab and an engineered derivative containing methionine residue M74, utilizing the principles of oxaziridine chemistry. Subsequently, these conjugates were functionalized with a modified DFO chelator incorporating dibenzocyclooctyne. The resulting DFO-WT and DFO-M74 conjugates were radiolabeled with generator-produced [ 68 Ga]Ga 3+ , to yield the novel PET radiotracers, [ 68 Ga]Ga-DFO-WT and [ 68 Ga]Ga-DFO-M74. In vitro and in vivo studies demonstrated that [ 68 Ga]Ga-DFO-M74 exhibited a higher affinity for HER2 receptors. Biodistribution studies in mice bearing orthotopic HER2-positive breast tumors revealed a higher uptake of [ 68 Ga]Ga-DFO-M74 in the tumor tissue, accompanied by rapid renal clearance, enabling clear delineation of tumors using PET imaging. Conversely, [ 68 Ga]Ga-DFO-WT exhibited lower uptake and inferior image contrast compared to [ 68 Ga]Ga-DFO-M74. Overall, the results demonstrate that the highly facile methionine-oxaziridine modification approach can be simply applied to the synthesis of stable and site-specifically modified radiolabeled antibody-chelator conjugates with favorable pharmacokinetics for PET imaging.