Selective ablation of TRA-1-60+ pluripotent stem cells suppresses tumor growth of prostate cancer.

Purpose: TRA-1-60 (TRA) is an established transcription factor of embryonic signaling and a well-known marker of pluripotency. It has been implicated in tumorigenesis and metastases, is not expressed in differentiated cells, which makes it an appealing biomarker for immunopositron emission tomography (immunoPET) imaging and radiopharmaceutical therapy (RPT). Herein, we explored the clinical implications of TRA in prostate cancer (PCa), examined the potential of TRA-targeted PET to specifically image TRA+ cancer stem cells (CSCs) and assessed response to the selective ablation of PCa CSCs using TRA-targeted RPT. Experimental Design: First, we assessed the relationship between TRA (PODXL) copy number alterations (CNA) and survival using publicly available patient databases. The anti-TRA antibody, Bstrongomab, was radiolabeled with Zr-89 or Lu-177 for immunoPET imaging and RPT in PCa xenografts. Radiosensitive tissues were collected to assess radiotoxicity while excised tumors were examined for pathologic treatment response. Results: Patients with tumors having high PODXL CNA exhibited poorer progression-free survival than those with low PODXL, suggesting that it plays an important role in tumor aggressiveness. TRA-targeted immunoPET imaging specifically imaged CSCs in DU-145 xenografts. Tumors treated with TRA RPT exhibited delayed growth and decreased proliferative activity, marked by Ki-67 immunohistochemistry. Aside from minor weight loss in select animals, no significant signs of radiotoxicity were observed in the kidneys or livers. Conclusions: We successfully demonstrated the clinical significance of TRA expression in human PCa, engineered and tested radiotheranostic agents to image and treat TRA+ prostate CSCs. Ablation of TRA+ CSCs blunted PCa growth. Future studies combining CSC ablation with standard treatment will be explored to achieve durable responses.

Perspectives on metals-based radioimmunotherapy (RIT): moving forward.

Radioimmunotherapy (RIT) is FDA-approved for the clinical management of liquid malignancies, however, its use for solid malignancies remains a challenge. The putative benefit of RIT lies in selective targeting of antigens expressed on the tumor surface using monoclonal antibodies, to systemically deliver cytotoxic radionuclides. The past several decades yielded dramatic improvements in the quality, quantity, recent commercial availability of alpha-, beta- and Auger Electron-emitting therapeutic radiometals. Investigators have created new or improved existing bifunctional chelators. These bifunctional chelators bind radiometals and can be coupled to antigen-specific antibodies. In this review, we discuss approaches to develop radiometal-based RITs, including the selection of radiometals, chelators and antibody platforms (i.e. full-length, F(ab')2, Fab, minibodies, diabodies, scFv-Fc and nanobodies). We cite examples of the performance of RIT in the clinic, describe challenges to its implementation, and offer insights to address gaps toward translation.

In vivo Imaging Technologies to Monitor the Immune System.

The past two decades have brought impressive advancements in immune modulation, particularly with the advent of both cancer immunotherapy and biologic therapeutics for inflammatory conditions. However, the dynamic nature of the immune response often complicates the assessment of therapeutic outcomes. Innovative imaging technologies are designed to bridge this gap and allow non-invasive visualization of immune cell presence and/or function in real time. A variety of anatomical and molecular imaging modalities have been applied for this purpose, with each option providing specific advantages and drawbacks. Anatomical methods including magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound provide sharp tissue resolution, which can be further enhanced with contrast agents, including super paramagnetic ions (for MRI) or nanobubbles (for ultrasound). Conjugation of the contrast material to an antibody allows for specific targeting of a cell population or protein of interest. Protein platforms including antibodies, cytokines, and receptor ligands are also popular choices as molecular imaging agents for positron emission tomography (PET), single-photon emission computerized tomography (SPECT), scintigraphy, and optical imaging. These tracers are tagged with either a radioisotope or fluorescent molecule for detection of the target. During the design process for immune-monitoring imaging tracers, it is important to consider any potential downstream physiologic impact. Antibodies may deplete the target cell population, trigger or inhibit receptor signaling, or neutralize the normal function(s) of soluble proteins. Alternatively, the use of cytokines or other ligands as tracers may stimulate their respective signaling pathways, even in low concentrations. As in vivo immune imaging is still in its infancy, this review aims to describe the modalities and immunologic targets that have thus far been explored, with the goal of promoting and guiding the future development and application of novel imaging technologies.

Removal of Fc Glycans from [89Zr]Zr-DFO-Anti-CD8 Prevents Peripheral Depletion of CD8+ T Cells.

The N-linked biantennary glycans on the heavy chain of immunoglobulin G (IgG) antibodies (mAbs) are instrumental in the recognition of the Fc region by Fc-gamma receptors (FcγR). In the case of full-length mAb-based imaging tracers targeting immune cell populations, these Fc:FcγR interactions can potentially deplete effector cells responsible for tumor clearance. To bypass this problem, we hypothesize that the enzymatic removal of the Fc glycans will disrupt Fc:FcγR interactions and spare tracer-targeted immune cells from depletion during immunopositron emission tomography (immunoPET) imaging. Herein, we compared the in vitro and in vivo properties of 89Zr-radiolabeled CD8-specific murine mAb (anti-CD8wt, clone 2.43), a well-known depleting mAb, and its deglycosylated counterpart (anti-CD8degly). Deglycosylation was achieved via enzymatic treatment with the peptide: N-glycosidase F (PNGaseF). Both anti-CD8wt and anti-CD8degly mAbs were conjugated to p-SCN-Bn-desferrioxamine (DFO) and labeled with 89Zr. Bindings of both DFO-conjugated mAbs to FcγR and CD8+ splenocytes were compared. In vivo imaging and distribution studies were conducted to examine the specificity and pharmacokinetics of the radioimmunoconjugates in tumor-naive and CT26 colorectal tumor-bearing mice. Ex vivo analysis of CD8+ T cell population in spleens and tumors obtained postimaging were measured via flow cytometry and qRT-PCR. The removal of the Fc glycans from anti-CD8wt was confirmed via SDS-PAGE. A reduction in FcγR interaction was exhibited by DFO-anti-CD8degly, while its binding to CD8 remained unchanged. Tissue distribution showed similar pharmacokinetics of [89Zr]Zr-DFO-anti-CD8degly and the wt radioimmunoconjugate. In vivo blocking studies further demonstrated retained specificity of the deglycosylated radiotracer for CD8. From the imaging studies, no difference in accumulation in both spleens and tumors was observed between both radiotracers. Results from the flow cytometry analysis confirmed depletion of CD8+ T cells in spleens of mice administered with DFO-anti-CD8wt, whereas an increase in CD8+ T cells was shown with DFO-anti-CD8degly. No statistically significant difference in tumor infiltrating CD8+ T cells was observed in cohorts administered with the probes when compared to control unmodulated mice. CD8 mRNA levels from excised tumors showed increased transcripts of the antigen in mice administered with [89Zr]Zr-DFO-anti-CD8degly compared to mice imaged with [89Zr]Zr-DFO-anti-CD8wt. In conclusion, the removal of Fc glycans offers a straightforward approach to develop full length antibody-based imaging probes specifically for detecting CD8+ immune molecules with no consequential depletion of their target cell population in peripheral tissues.

Detecting TRA-1-60 in Cancer via a Novel Zr-89 Labeled ImmunoPET Imaging Agent.

TRA-1-60 (TRA) is a cell-surface antigen implicated in drug resistance, relapse, and recurrence. Its expression has been reported in breast, prostate, pancreatic, ovarian tumors, and follicular lymphoma, which paved the development of the therapeutic antibody, Bstrongomab (Bsg), and its drug conjugates. Because patient selection is critical to achieve clinical benefit, a noninvasive imaging agent to select TRA+ lesions in patients is needed. Herein, we report the development of the immunopositron emission tomography (immunoPET) radiotracer 89Zr-radiolabeled Bsg and its potential to delineate TRA+ tumors. Bsg was conjugated to the bifunctional chelator desferrioxamine (DFO) and radiolabeled with [89Zr]Zr-oxalate. [89Zr]Zr-DFO-Bsg was characterized in vitro and evaluated in vivo for uptake and specificity in high and low TRA-expressing BxPC-3 pancreatic and PC-3 prostate cancer models, respectively. Uptake was compared against [89Zr]Zr-DFO-IgG, a nonspecific control radiotracer. Immunohistochemical (IHC) staining of patient cancer tissues using Bsg was performed to explore its clinical significance. A specific activity of 0.18 ± 0.01 GBq/mg (4.8 ± 0.3 mCi/mg) was obtained for [89Zr]Zr-DFO-Bsg. BxPC-3 xenografts exhibited three-fold higher radiotracer uptake compared to [89Zr]Zr-DFO-IgG. Competitive saturation studies using BxPC-3 xenografts further confirmed tracer specificity. The TRA-specific probe had lower accumulation in PC-3 xenografts. Ex vivo autoradiographs correlated with TRA expression from the histopathology of the resected tumor xenografts. Additionally, patient cancer tissues demonstrated positive staining with Bsg with metastatic lesions exhibiting the highest staining. This study demonstrates the potential of [89Zr]Zr-DFO-Bsg as an imaging agent for noninvasive detection of TRA+ tumors.

Systemically Administered Plant Recombinant Holo-Intrinsic Factor Targets the Liver and is not Affected by Endogenous B12 levels.

Precision targeting imaging agents and/or treatment agents to select cells or organs in the body remains a significant need and is an area of intense research. It has been hypothesized that the vitamin B12 (B12) dietary pathway, or components thereof, may be exploitable in this area. The question of whether gastric Intrinsic factor (IF), critical for B12 absorption in the GI tract via the cubilin receptor, could be used as a targeting moiety for the cubilin receptor systemically, has not been investigated. Cubilin is the only known receptor for holo-IF and is found primarily in the kidney and ear (outside of the ileum of the GI) offering significant scope for specific targeting. We utilized plant derived human gastric IF in fluorescent cell and PET based in vivo imaging and biodistribution studies and demonstrated that plant derived IF primarily targets the liver, likely a consequence of the unique glycosylation profile of the IF, and is not affected by endogenous B12 levels.

Phosphorylation of the type II transmembrane serine protease, TMPRSS13, in hepatocyte growth factor activator inhibitor-1 and -2-mediated cell-surface localization.

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Although various TTSPs have been characterized in detail biochemically and functionally, the basic properties of TMPRSS13 remain unclear. Here, we investigate the activation, inhibition, post-translational modification, and localization of TMPRSS13. We show that TMPRSS13 is a glycosylated, active protease and that its own proteolytic activity mediates zymogen cleavage. Full-length, active TMPRSS13 exhibits impaired cell-surface expression in the absence of the cognate Kunitz-type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 or HAI-2. Concomitant presence of TMPRSS13 with either HAI-1 or -2 mediates phosphorylation of residues in the intracellular domain of the protease, and it coincides with efficient transport of the protease to the cell surface and its subsequent shedding. Cell-surface labeling experiments indicate that the dominant form of TMPRSS13 on the cell surface is phosphorylated, whereas intracellular TMPRSS13 is predominantly non-phosphorylated. These data provide novel insight into the cellular properties of TMPRSS13 and highlight phosphorylation of TMPRSS13 as a novel post-translational modification of this TTSP family member and potentially other members of this family of proteases.