Kallikreins are a family of serine proteases with a range of tissue-specific and essential proteolytic functions. Among the best studied are the prostate tissue-specific KLK2 and KLK3 genes and their secreted protease products, human kallikrein 2, hk2, and prostate-specific antigen (PSA). Members of the so-called classic kallikreins, these highly active trypsin-like serine proteases play established roles in human reproduction. Both hK2 and PSA expression is regulated by the androgen receptor which has a fundamental role in prostate tissue development and progression of disease. This feature, combined with the ability to sensitively detect different forms of these proteins in blood and biopsies, result in a crucially important biomarker for the presence and recurrence of cancer. Emerging evidence has begun to suggest a role for these kallikreins in critical vascular events. This review discusses the established and developing biological roles of hK2 and PSA, as well as the historical and advanced use of their detection to accurately and non-invasively detect and guide treatment of prostatic disease.

Targeted alpha therapy is an emerging strategy for the treatment of disseminated cancer. [²²³Ra]RaCl₂ is the only clinically approved alpha particle-emitting drug, and it is used to treat castrate-resistant prostate cancer bone metastases, to which [²²³Ra]Ra²⁺ localizes. To specifically direct [²²³Ra]Ra²⁺ to non-osseous disease sites, chelation and conjugation to a cancer-targeting moiety is necessary. Although previous efforts to stably chelate [²²³Ra]Ra²⁺ for this purpose have had limited success, here we report a biologically stable radiocomplex with the 18-membered macrocyclic chelator macropa. Quantitative labeling of macropa with [²²³Ra]Ra²⁺ was accomplished within 5 min at room temperature with a radiolabeling efficiency of >95%, representing a significant advancement over conventional chelators such as DOTA and EDTA, which were unable to completely complex [²²³Ra]Ra²⁺ under these conditions. [²²³Ra][Ra(macropa)] was highly stable in human serum and exhibited dramatically reduced bone and spleen uptake in mice in comparison to bone-targeted [²²³Ra]RaCl₂, signifying that [²²³Ra][Ra(macropa)] remains intact in vivo. Upon conjugation of macropa to a single amino acid β-alanine as well as to the prostate-specific membrane antigen-targeting peptide DUPA, both constructs retained high affinity for ²²³Ra, complexing >95% of Ra²⁺ in solution. Furthermore, [²²³Ra][Ra(macropa-β-alanine)] was rapidly cleared from mice and showed low ²²³Ra bone absorption, indicating that this conjugate is stable under biological conditions. Unexpectedly, this stability was lost upon conjugation of macropa to DUPA, which suggests a role of targeting vectors in complex stability in vivo for this system. Nonetheless, our successful demonstration of efficient radiolabeling of the β-alanine conjugate with ²²³Ra and its subsequent stability in vivo establishes for the first time the possibility of delivering [²²³Ra]Ra²⁺ to metastases outside of the bone using functionalized chelators, marking a significant expansion of the therapeutic utility of this radiometal in the clinic.

Purpose

The impact of androgen receptor (AR) activity in breast cancer biology is unclear. We characterized and tested a novel therapy to an AR-governed target in breast cancer.Experimental Design: We evaluated the expression of prototypical AR gene products human kallikrein 2 (hK2) and PSA in breast cancer models. We screened 13 well-characterized breast cancer cell lines for hK2 and PSA production upon in vitro hormone stimulation by testosterone [dihydrotestosterone (DHT)]. AR-positive lines were further evaluated by exposure to estrogen (17β-Estradiol) and the synthetic progestin D-Norgestrel. We then evaluated an anti-hK2-targeted radiotherapy platform (hu11B6), labeled with alpha (α)-particle emitting Actinium-225, to specifically treat AR-expressing breast cancer xenografts under hormone stimulation.

Results

D-Norgestrel and DHT activated the AR pathway, while 17β-Estradiol did not. Competitive binding for AR protein showed similar affinity between DHT and D-Norgestrel, indicating direct AR-ligand interaction. In vivo production of hK2 was sufficient to achieve site-specific delivery of therapeutic radionuclide to tumor tissue at >20-fold over background muscle uptake; effecting long-term local tumor control.

Conclusions

[²²⁵Ac]hu11B6 targeted radiotherapy was potentiated by DHT and by D-Norgestrel in murine xenograft models of breast cancer. AR activity in breast cancer correlates with kallikrein-related peptidase-2 and can be activated by D-Norgestrel, a common contraceptive, and AR induction can be harnessed for hK2-targeted breast cancer α-emitter radiotherapy.

PYTA is demonstrated to rapidly and stably complex 225Ac, 177Lu, 111In, and 44Sc, a “superfecta” of complementary but chemically distinct radiometals for targeted theranostic applications.

Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans.

Bacterial biofilm infections of implantable medical devices decrease the effectiveness of antibiotics, creating difficult-to-treat chronic infections. Prosthetic joint infections (PJI) are particularly problematic because they require prolonged antibiotic courses and reoperations to remove and replace the infected prostheses. Current models to study PJI focus on Gram-positive bacteria, but Gram-negative PJI (GN-PJI) are increasingly common and are often more difficult to treat, with worse clinical outcomes. Herein, we sought to develop a mouse model of GN-PJI to investigate the pathogenesis of these infections and identify potential therapeutic targets. An orthopedic-grade titanium implant was surgically placed in the femurs of mice, followed by infection of the knee joint with Pseudomonas aeruginosa or Escherichia coli. We found that in vitro biofilm-producing activity was associated with the development of an in vivo orthopedic implant infection characterized by bacterial infection of the bone/joint tissue, biofilm formation on the implants, reactive bone changes, and inflammatory immune cell infiltrates. In addition, a bispecific antibody targeting P. aeruginosa virulence factors (PcrV and Psl exopolysaccharide) reduced the bacterial burden in vivo. Taken together, our findings provide a preclinical model of GN-PJI and suggest the therapeutic potential of targeting biofilm-associated antigens.

Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, ⁸⁹Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.