Kevin J. H. Allen

Background: the primary challenge with implant infections is the formation of biofilm, which harbors dormant bacteria that reduce the effectiveness of antibiotics and amplify antibiotic resistance, exacerbating the global antimicrobial resistance crisis. A potential novel treatment strategy is radioimmunotherapy, which uses antibodies linked to radioisotopes to deliver targeted radiation to the bacteria and biofilm. We describe the first in vivo use of targeted radiation therapy, employing Actinium-225 (α-radiation) and Lutetium-177 (β-radiation) labeled antibodies to treat a Staphylococcus aureus biofilm-associated intramedullary implant infection. Untargeted radiation in the form of unbound radionuclide treatment was also evaluated. Methods: to assess therapeutic efficacy, bacterial counts were performed on implant and surrounding bone after seven days of follow-up. Biodistribution was evaluated using SPECT/CT and ex vivo gamma counting. Results: radioimmunotherapy using an antibody against wall teichoic acid which was labeled with Actinium-225 and Lutetium-177 achieved bacterial reductions between 45% and 93% on the implant and surrounding bone. Surprisingly, a similar antimicrobial effect was observed with unbound Actinium-225 treatment reducing the bacterial load by 80% on the implant and 98% in the surrounding bone. Indications of maximum tolerated dose (MTD) with Lutetium-177 labeled antibodies were observed through hepatic and renal function evaluations. Conclusions: These results should be interpreted in the context of the study’s constraints, particularly the limited animal sample size. Nonetheless, the results suggest that in vivo applied radiation may help reduce a biofilm-associated infection at the implant site as well as in the surrounding bone. These findings encourage further investigation into the use of targeted and non-targeted radiation, potentially combined with antibiotics, to develop effective strategies for eradicating biofilm-associated implant infections.

We previously found that chronic adenosine A1 receptor stimulation with N⁶-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson’s disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood–brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioral abnormalities, and neurodegeneration in our rodent model. Behavioral tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the ¹⁸F-labeled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-β-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein “loop” conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The ¹⁸F-labeled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson’s disease therapy.

We previously found that chronic adenosine A1 receptor stimulation with N6-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson’s disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood-brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioural abnormalities, and neurodegeneration in our rodent model. Behavioural tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the 18F-labelled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-β-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein “loop” conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The 18F-labelled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson’s disease therapy.

Metastatic melanoma is a deadly disease that claims thousands of lives each year despite the introduction of several immunotherapeutic agents into the clinic over the past decade, inspiring the development of novel therapeutics and the exploration of combination therapies. Our investigations target melanin pigment with melanin-specific radiolabeled antibodies as a strategy to treat metastatic melanoma. In this study, a theranostic approach was applied by first labeling a chimeric antibody targeting melanin, c8C3, with the SPECT radionuclide ²⁰³Pb for microSPECT/CT imaging of C57Bl6 mice bearing B16-F10 melanoma tumors. Imaging was followed by radioimmunotherapy (RIT), whereby the c8C3 antibody is radiolabeled with a ²¹²Pb/²¹²Bi “in vivo generator”, which emits cytotoxic alpha particles. Using microSPECT/CT, we collected sequential images of B16-F10 murine tumors to investigate antibody biodistribution. Treatment with the ²¹²Pb/²¹²Bi-labeled c8C3 antibody demonstrated a dose-response in tumor growth rate in the 5–10 µCi dose range when compared to the untreated and radiolabeled control antibody and a significant prolongation in survival. No hematologic or systemic toxicity of the treatment was observed. However, administration of higher doses resulted in a biphasic tumor dose response, with the efficacy of treatment decreasing when the administered doses exceeded 10 µCi. These results underline the need for more pre-clinical investigation of targeting melanin with ²¹²Pb-labeled antibodies before the clinical utility of such an approach can be assessed.

Background: Osteosarcoma (OS) represents the most common primary bone tumor in humans and in companion dogs, being practically phenotypically identical. There is a need for effective treatments to extend the survival of patients with OS. Here, we examine the dosimetry in beagle dogs and cross-reactivity with human tissues of a novel human antibody, IF3, that targets the insulin growth factor receptor type 2 (IGF2R), which is overexpressed on OS cells, making it a candidate for radioimmunotherapy of OS. Methods: [⁸⁹Zr]Zr-DFO-IF3 was injected into three healthy beagle dogs. PET/CT was conducted at 4, 24, 48, and 72 h. RAPID analysis was used to determine the dosimetry of [¹⁷⁷Lu]Lu-CHXA”-IF3 for a clinical trial in companion dogs with OS. IF3 antibody was biotinylated, and a multitude of human tissues were assessed with immunohistochemistry. Results: PET/CT revealed that only the liver, bone marrow, and adrenal glands had high uptake. Clearance was initially through renal and hepatobiliary excretion in the first 72 h followed by primarily physical decay. RAPID analysis showed bone marrow to be the dose-limiting organ with a therapeutic range for ¹⁷⁷Lu calculated to be 0.487–0.583 GBq. Immunohistochemistry demonstrated the absence of IGF2R expression on the surface of healthy human cells, thus suggesting that radioimmunotherapy with [¹⁷⁷Lu]Lu-CHXA”-IF3 will be well tolerated. Conclusions: Image-based dosimetry has defined a safe therapeutic range for canine clinical trials, while immunohistochemistry has suggested that the antibody will not cross-react with healthy human tissues.

Objective: Positron emission tomography (PET) imaging is a powerful non-invasive method to determine the in vivo behavior of biomolecules. Determining biodistribution and pharmacokinetic (PK) properties of targeted therapeutics can enable a better understanding of in vivo drug mechanisms such as tumor uptake, off target accumulation and clearance. Zirconium-89 (⁸⁹Zr) is a readily available tetravalent PET-enabling radiometal that has been used to evaluate the biodistribution and PK of monoclonal antibodies. In the current study, we performed in vitro and in vivo characterization of ⁸⁹Zr-lintuzumab, a radiolabeled anti-CD33 antibody, as a model to evaluate the in vivo binding properties in preclinical models of AML. Methods: Lintuzumab was conjugated to p-SCN-Bn-deferoxamine (DFO) and labeled with ⁸⁹Zr using a 5:1 µCi:µg specific activity at 37 °C for 1h. The biological activity of ⁸⁹Zr-lintuzumab was evaluated in a panel of CD33 positive cells using flow cytometry. Fox Chase SCID mice were injected with 2 × 10⁶ OCI-AML3 cells into the right flank. After 12 days, a cohort of mice (n = 4) were injected with ⁸⁹Zr-lintuzumab via tail vein. PET/CT scans of mice were acquired on days 1, 2, 3 and 7 post ⁸⁹Zr-lintuzumab injection. To demonstrate ⁸⁹Zr-lintuzumab specific binding to CD33 expressing tumors in vivo, a blocking study was performed. This cohort of mice (n = 4) was injected with native lintuzumab and 24 h later ⁸⁹Zr-lintuzumab was administered. This group was imaged 3 and 7 days after injection of ⁸⁹Zr-lintuzumab. A full ex vivo biodistribution study on both cohorts was performed on day 7. The results from the PET image and ex vivo biodistribution studies were compared. Results: Lintuzumab was successfully radiolabeled with ⁸⁹Zr resulting in a 99% radiochemical yield. The ⁸⁹Zr-lintuzumab radioconjugate specifically binds CD33 positive cells in a similar manner to native lintuzumab as observed by flow cytometry. PET imaging revealed high accumulation of ⁸⁹Zr-lintuzumab in OCI-AML3 tumors within 24h post-injection of the radioconjugate. The ⁸⁹Zr-lintuzumab high tumor uptake remains for up to 7 days. Tumor analysis of the PET data using volume of interest (VOI) showed significant blocking of ⁸⁹Zr-lintuzumab in the group pre-treated with native lintuzumab (pre-blocked group), thus indicating specific targeting of CD33 on OCI-AML3 cells in vivo. The tumor uptake findings from the PET imaging study are in agreement with those from the ex vivo biodistribution results. Conclusions: PET imaging of ⁸⁹Zr-lintuzumab shows high specific uptake in CD33 positive human OCI-AML3 tumors. The results from the image study agree with the observations from the ex vivo biodistribution study. Our findings collectively suggest that PET imaging using ⁸⁹Zr-lintuzumab could be a powerful drug development tool to evaluate binding properties of anti-CD33 monoclonal antibodies in preclinical cancer models.

Nearly 100,000 individuals are expected to be diagnosed with melanoma in the United States in 2022. Treatment options for late-stage metastatic disease up until the 2010s were few and offered only slight improvement to the overall survival. The introduction of B-RAF inhibitors and anti-CTLA4 and anti-PD-1/PD-L1 immunotherapies into standard of care brought measurable increases in the overall survival across all stages of melanoma. Despite the improvement in the survival statistics, patients treated with targeted therapies and immunotherapies are subject to very serious side effects, the development of drug resistance, and the high costs of treatment. This leaves room for the development of novel approaches as well as for the exploration of novel combination therapies for the treatment of metastatic melanoma. One such approach is targeting melanin pigment with radionuclide therapy. Advances in melanin-targeting radionuclide therapy of melanoma can be viewed from two spheres: (1) radioimmunotherapy (RIT) and (2) radiolabeled small molecules. The investigation of mechanisms of the action and efficacy of targeting melanin in melanoma treatment by RIT points to the involvement of the immune system such as complement dependent cytotoxicity. The combination of RIT with immunotherapy presents synergistic killing in mouse melanoma models. The field of radiolabeled small molecules is focused on radioiodinated compounds that have the ability to cross the cellular membranes to access intracellular melanin and can be applied in both therapy and imaging as theranostics. Clinical applications of targeting melanin with radionuclide therapies have produced encouraging results and clinical work is on-going. Continued work on targeting melanin with radionuclide therapy as a monotherapy, or possibly in combination with standard of care agents, has the potential to strengthen the current treatment options for melanoma patients.

Background: Osteosarcoma (OS) has an overall patient survival rate of ~70% with no significant improvements in the last two decades, and novel effective treatments are needed. OS in companion dogs is phenotypically close to human OS, which makes a comparative oncology approach to developing new treatments for OS very attractive. We have recently created a novel human antibody, IF3 to IGF2R, which binds to this receptor on both human and canine OS tumors. Here, we evaluated the efficacy and safety of radioimmunotherapy with ¹⁷⁷Lu-labeled IF3 of mice bearing canine-patient-derived tumors and performed canine and human dosimetry calculations. Methods: Biodistribution and microSPECT/CT imaging with ¹¹¹In-IF3 was performed in mice bearing canine OS Gracie tumors, and canine and human dosimetry calculations were performed based on these results. RIT of Gracie-tumor-bearing mice was completed with ¹⁷⁷Lu-IF3. Results: Biodistribution and imaging showed a high uptake of ¹¹¹In-IF3 in the tumor and spleen. Dosimetry identified the tumor, spleen and pancreas as the organs with the highest uptake. RIT was very effective in abrogating tumor growth in mice with some spleen-associated toxicity. Conclusions: These results demonstrate that RIT with ¹⁷⁷Lu-IF3 targeting IGF2R on experimental canine OS tumors effectively decreases tumor growth. However, because of the limitations of murine models, careful evaluation of the possible toxicity of this treatment should be performed via nuclear imaging and image-based dosimetry in healthy dogs before clinical trials in companion dogs with OS can be attempted.

Etiological and genetic drivers of osteosarcoma (OS) are not well studied and vary from one tumor to another; making it challenging to pursue conventional targeted therapy. Recent studies have shown that cation independent mannose-6-phosphate/insulin-like growth factor-2 receptor (IGF2R) is consistently overexpressed in almost all of standard and patient-derived OS cell lines, making it an ideal therapeutic target for development of antibody-based drugs. Monoclonal antibodies, targeting IGF2R, can be conjugated with alpha- or beta-emitter radionuclides to deliver cytocidal doses of radiation to target IGF2R expression in OS. This approach known as radioimmunotherapy (RIT) can therefore be developed as a novel treatment for OS. In addition, OS is one of the common cancers in companion dogs and very closely resembles human OS in clinical presentation and molecular aberrations. In this study, we have developed human antibodies that cross-react with similar affinities to IGF2R proteins of human, canine and murine origin. We used naïve and synthetic antibody Fab-format phage display libraries to develop antibodies to a conserved region on IGF2R. The generated antibodies were radiolabeled and characterized in vitro and in vivo using human and canine OS patient-derived tumors in SCID mouse models. We demonstrate specific binding to IGF2R and tumor uptake in these models, as well as binding to tumor tissue of canine OS patients, making these antibodies suitable for further development of RIT for OS

Melanoma incidence continues to rise, and while therapeutic approaches for early stage cases are effective, metastatic melanoma continues to be associated with high mortality. Immune checkpoint blockade (ICB) has demonstrated clinical success with approved drugs in cohorts of patients with metastatic melanoma and targeted radionuclide therapy strategies showed promise in several clinical trials against various cancers including metastatic melanoma. This led our group to investigate the combination of these two treatments which could be potentially offered to patients with metastatic melanoma not responsive to ICB alone. Previously, we have demonstrated that a combination of humanized anti-melanin antibody conjugated to 213Bismuth and anti-PD-1 ICB reduced tumor growth and increased survival in the Cloudman S91 murine melanoma DBA/2 mouse model. In the current study, we sought to improve the tumoricidal effect by using the long-lived radionuclides 177Lutetium and 225Actinium. Male Cloudman S91-bearing DBA/2 mice were treated intraperitoneally with PBS (Sham), unlabeled antibody to melanin, anti-PD-1 ICB, 177Lutetium or 225Actinium RIT, or a combination of ICB and RIT. Treatment with anti-PD-1 alone or low-dose 177Lutetium RIT alone resulted in modest tumor reduction, while their combination significantly reduced tumor growth and increased survival, suggesting synergy. 225Actinium RIT, alone or in combination with ICB, showed no therapeutic benefit, suggesting that the two radionuclides with different energetic properties work in distinct ways. We did not detect an increase in tumor-infiltrating T cells in the tumor microenvironment, which suggests the involvement of alternative mechanisms that improve the effect of combination therapy beyond that observed in the single therapies.