Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo.
ABSTRACT: The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS22.214.171.124) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.
Project description:The proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole-body T lymphocyte dynamics noninvasively in vivo, we generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for (89)Zr-immuno-PET detection of helper and cytotoxic T cell populations.Anti-CD4 and -CD8 cDbs were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cDbs were site-specifically conjugated to maleimide-desferrioxamine for (89)Zr radiolabeling and subsequent small-animal PET/CT acquisition and ex vivo biodistribution in both wild-type mice and a model of hematopoietic stem cell (HSC) transplantation.Immuno-PET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wild-type mice, with specificity confirmed through in vivo blocking and depletion studies. Subsequently, a murine model of HSC transplantation demonstrated successful in vivo detection of T cell repopulation at 2, 4, and 8 wk after HSC transplantation using the (89)Zr-radiolabeled anti-CD4 and -CD8 cDbs.These newly developed anti-CD4 and -CD8 immuno-PET reagents represent a powerful resource to monitor T cell expansion, localization, and novel engraftment protocols. Future potential applications of T cell-targeted immuno-PET include monitoring immune cell subsets in response to immunotherapy, autoimmunity, and lymphoproliferative disorders, contributing overall to preclinical immune cell monitoring.
Project description:Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g., copper-64, (64)Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of (64)Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10-3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g., pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with (64)Cu.
Project description:Copper-64 (<sup>64</sup>Cu)-labeled antibody fragments such as Fab are useful for molecular imaging (immuno-PET) and radioimmunotherapy. However, these fragments cause high and persistent localization of radioactivity in the kidneys after injection. To solve this problem, this study assessed the applicability of a molecular design to <sup>64</sup>Cu, which reduces renal radioactivity levels by liberating a urinary excretory radiometabolite from antibody fragments at the renal brush border membrane (BBM). Since 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) forms a stable complex with Cu, NOTA-conjugated Met-Val-Lys-maleimide (NOTA-MVK-Mal), which is a radio-gallium labeling agent for antibody fragments, was evaluated for applicability to <sup>64</sup>Cu. The MVK linkage was recognized by the BBM enzymes to liberate [<sup>64</sup>Cu]Cu-NOTA-Met although the recognition of the MVK sequence for the [<sup>64</sup>Cu]Cu-NOTA-MVK derivative was reduced compared with that of its [<sup>67</sup>Ga]Ga-counterpart, probably due to the difference in the charge of the metal-NOTA complexes. When injected into mice, [<sup>64</sup>Cu]Cu-NOTA-MVK-Fab resulted in similar renal radioactivity levels to the <sup>67</sup>Ga-labeled counterpart. In addition, [<sup>64</sup>Cu]Cu-NOTA-MVK-Fab resulted in lower renal radioactivity levels than those from <sup>64</sup>Cu-labeled Fab using a conventional method, without a reduction in the tumor radioactivity levels. These findings indicate that our approach to reducing renal radioactivity levels by liberating a radiolabeled compound from antibody fragments at the renal BBM for urinary excretion is applicable to <sup>64</sup>Cu-labeled antibody fragments and useful for immuno-PET and radioimmunotherapy.
Project description:Development of multifunctional and well-dispersed hollow mesoporous silica nanoparticles (HMSNs) for tumor vasculature targeted drug delivery and PET imaging.Amine functionalized HMSNs (150-250 nm) were conjugated with a macrocyclic chelator, (S)-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triaceticacid (NOTA), PEGylated and loaded with antiangiogenesis drug, Sunitinib. Cyclo(Arg-Gly-Asp-D-Tyr-Lys) (cRGDyK) peptide was attached to the nanoconjugate and radiolabeled with (64)Cu for PET imaging.(64)Cu-NOTA-HMSN-PEG-cRGDyK exhibited integrin-specific uptake both in vitro and in vivo. PET results indicated approximately 8% ID/g uptake of targeted nanoconjugates in U87MG tumors, which correlated well with ex vivo and histological analyses. Enhanced tumor-targeted delivery of sunitinib was also observed.We successfully developed tumor vasculature targeted HMSNs for PET imaging and image-guided drug delivery.
Project description:Macrocyclic chelators have been widely employed in the realm of nanoparticle-based positron emission tomography (PET) imaging, whereas its accuracy remains questionable. Here, we found that 64 Cu can be intrinsically labeled onto nanographene based on interactions between Cu and the ??electrons of graphene without the need of chelator conjugation, providing a promising alternative radiolabeling approach that maintains the native in?vivo pharmacokinetics of the nanoparticles. Due to abundant ??bonds, reduced graphene oxide (RGO) exhibited significantly higher labeling efficiency in comparison with graphene oxide (GO) and exhibited excellent radiostability in?vivo. More importantly, nonspecific attachment of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) on nanographene was observed, which revealed that chelator-mediated nanoparticle-based PET imaging has its inherent drawbacks and can possibly lead to erroneous imaging results in?vivo.
Project description:Purpose: Among the treatment options for pancreatic ductal adenocarcinoma (PDAC) are antibodies against the programmed cell death receptor 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway. Positron emission tomography (PET) has been successfully used to assess PD-1/PD-L1 signaling in subcutaneous tumor models, but orthotopic tumor models are increasingly being recognized as a better option to accurately recapitulate human disease. However, when PET radiotracers have high uptake in the liver and spleen, it can obscure signals from the adjacent pancreas, making visualization of the response in orthotopic pancreatic tumors technically challenging. In this study, we first investigated the impact of radioisotope chelators on the biodistribution of 64Cu-labeled anti-PD-1 and anti-PD-L1 antibodies and compared the distribution profiles of anti-PD-1 and anti-PD-L1 antibodies. We then tested the hypothesis that co-injection of unlabeled antibodies reduces uptake of 64Cu-labeled anti-PD-L1 antibodies in the spleen and thereby permits accurate delineation of orthotopic pancreatic tumors in mice. Procedures: We established subcutaneous and orthotopic mouse models of PDAC using KRAS* murine pancreatic cancer cells with a doxycycline-inducible mutation of KRASG12D. We then (1) compared the biodistribution of 64Cu-labeled anti-PD-1 with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) and 2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) used as the chelators in the orthotopic model; (2) compared the biodistribution of [64Cu]Cu-NOTA-anti-PD-1 and [64Cu]Cu-NOTA-anti-PD-L1 in the orthotopic model; and (3) imaged subcutaneous and orthotopic KRAS* tumors with [64Cu]Cu-NOTA-anti-PD-L1 with and without co-injection of unlabeled anti-PD-L1 as the blocking agent. Results: [64Cu]Cu-NOTA-anti-PD-L1 was a promising imaging probe. By co-injection of an excess of unlabeled anti-PD-L1, background signals of [64Cu]Cu-NOTA-anti-PD-L1 from the spleen were significantly reduced, leading to a clear delineation of orthotopic pancreatic tumors. Conclusions: Co-injection with unlabeled anti-PD-L1 is a useful method for PET imaging of PD-L1 expression in orthotopic pancreatic cancer models.
Project description:The potential of PACE4 as a pharmacological target in prostate cancer has been demonstrated as this proprotein convertase is strongly overexpressed in human prostate cancer tissues and its inhibition, using molecular or pharmacological approaches, results in reduced cell proliferation and tumor progression in mouse tumor xenograft models. We developed a PACE4 high-affinity peptide inhibitor, namely, the multi-leucine (ML), and sought to determine whether this peptide could be exploited for the targeting of prostate cancer for diagnostic or molecular imaging purposes. We conjugated a bifunctional chelator 1,4,7-triazacyclononane-1,4,7- triacetic acid (NOTA) to the ML peptide for copper-64 ((64)Cu) labeling and positron emission tomography (PET)- based prostate cancer detection. Enzyme kinetic assays against recombinant PACE4 showed that the NOTA-modified ML peptide displays identical inhibitory properties compared to the unmodified peptide. In vivo biodistribution of the (64)Cu/NOTA-ML peptide evaluated in athymic nude mice bearing xenografts of two human prostate carcinoma cell lines showed a rapid and high uptake in PACE4-expressing LNCaP tumor at an early time point and in PACE4-rich organs. Co-injection of unlabeled peptide confirmed that tumor uptake was target-specific. PACE4-negative tumors displayed no tracer uptake 15 minutes after injection, while the kidneys, demonstrated high uptake due to rapid renal clearance of the peptide. The present study supports the feasibility of using a (64)Cu/NOTA-ML peptide for PACE4-targeted prostate cancer detection and PACE4 status determination by PET imaging but also provides evidence that ML inhibitor-based drugs would readily reach tumor sites under in vivo conditions for pharmacological intervention or targeted radiation therapy.
Project description:<h4>Purpose</h4>Glioblastoma (GBM) is the most common malignant brain tumor in adults. Various immunotherapeutic approaches to improve patient survival are being developed, but the molecular mechanisms of immunotherapy resistance are currently unknown. Here, we explored the ability of a humanized radiolabeled CD8-targeted minibody to noninvasively quantify tumor-infiltrating CD8-positive (CD8<sup>+</sup>) T cells using PET.<h4>Experimental design</h4>We generated a peripheral blood mononuclear cell (PBMC) humanized immune system (HIS) mouse model and quantified the absolute number of CD8<sup>+</sup> T cells by flow cytometry relative to the [<sup>64</sup>Cu]Cu-NOTA-anti-CD8 PET signal. To evaluate a patient-derived orthotopic GBM HIS model, we intracranially injected cells into NOG mice, humanized cohorts with multiple HLA-matched PBMC donors, and quantified CD8<sup>+</sup> tumor-infiltrating lymphocytes by IHC. To determine whether [<sup>64</sup>Cu]Cu-NOTA-anti-CD8 images brain parenchymal T-cell infiltrate in GBM tumors, we performed PET and autoradiography and subsequently stained serial sections of brain tumor tissue by IHC for CD8<sup>+</sup> T cells.<h4>Results</h4>Nontumor-bearing NOG mice injected with human PBMCs showed prominent [<sup>64</sup>Cu]Cu-NOTA-anti-CD8 uptake in the spleen and minimal radiotracer localization to the normal brain. NOG mice harboring intracranial human GBMs yielded high-resolution PET images of tumor-infiltrating CD8<sup>+</sup> T cells. Radiotracer retention correlated with CD8<sup>+</sup> T-cell numbers in spleen and tumor tissue. Our study demonstrates the ability of [<sup>64</sup>Cu]Cu-NOTA-anti-CD8 PET to quantify peripheral and tumor-infiltrating CD8<sup>+</sup> T cells in brain tumors.<h4>Conclusions</h4>Human CD8<sup>+</sup> T cells infiltrate an orthotopic GBM in a donor-dependent manner. Furthermore, [<sup>64</sup>Cu]Cu-NOTA-anti-CD8 quantitatively images both peripheral and brain parenchymal human CD8<sup>+</sup> T cells.
Project description:The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific ?PD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane- N, N', N?-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, 64Cu-NOTA-?PD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.
Project description:The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA.Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results.NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies.(64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA.