Development and therapeutic evaluation of 5D3(CC-MLN8237)3.2 antibody-theranostic conjugates for PSMA-positive prostate cancer therapy.

Prostate cancer (PC) is an aggressive cancer that can progress rapidly and eventually become castrate-resistant prostate cancer (CRPC). Stage IV metastatic castrate-resistant prostate cancer (mCRPC) is an incurable late-stage cancer type with a low 5-year overall survival rate. Targeted therapeutics such as antibody-drug conjugates (ADCs) based on high-affinity monoclonal antibodies and potent drugs conjugated via smart linkers are being developed for PC management. Conjugating further with in vitro or in vivo imaging agents, ADCs can be used as antibody-theranostic conjugates (ATCs) for diagnostic and image-guided drug delivery. In this study, we have developed a novel ATC for PSMA (+) PC therapy utilizing (a) anti-PSMA 5D3 mAb, (b) Aurora A kinase inhibitor, MLN8237, and (c) for the first time using tetrazine (Tz) and trans-cyclooctene (TCO) click chemistry-based conjugation linker (CC linker) in ADC development. The resulting 5D3(CC-MLN8237)3.2 was labeled with suitable fluorophores for in vitro and in vivo imaging. The products were characterized by SDS-PAGE, MALDI-TOF, and DLS and evaluated in vitro by optical imaging, flow cytometry, and WST-8 assay for cytotoxicity in PSMA (+/-) cells. Therapeutic efficacy was determined in human PC xenograft mouse models following a designed treatment schedule. After the treatment study animals were euthanized, and toxicological studies, complete blood count (CBC), blood clinical chemistry analysis, and H&E staining of vital organs were conducted to determine side effects and systemic toxicities. The IC50 values of 5D3(CC-MLN8237)3.2-AF488 in PSMA (+) PC3-PIP and PMSA (-) PC3-Flu cells are 8.17 nM and 161.9 nM, respectively. Pure MLN8237 shows 736.9 nM and 873.4 nM IC50 values for PC3-PIP and PC3-Flu cells, respectively. In vivo study in human xenograft mouse models confirmed high therapeutic efficacy of 5D3(CC-MLN8237)3.2-CF750 with significant control of PSMA (+) tumor growth with minimal systemic toxicity in the treated group compared to PSMA (-) treated and untreated groups. Approximately 70% of PSMA (+) PC3-PIP tumors did not exceed the threshold of the tumor size in the surrogate Kaplan-Meyer analysis. The novel ATC successfully controlled the growth of PSMA (+) tumors in preclinical settings with minimal systemic toxicities. The therapeutic efficacy and favorable safety profile of novel 5D3(CC-MLN8237)3.2 ATC demonstrates their potential use as a theranostic against aggressive PC.

Therapeutic efficacy of an alpha-particle emitter labeled anti-GD2 humanized antibody against osteosarcoma-a proof of concept study.

PURPOSE: Current treatments for osteosarcoma (OS) have a poor prognosis, particularly for patients with metastasis and recurrence, underscoring an urgent need for new targeted therapies to improve survival. Targeted alpha-particle therapy selectively delivers cytotoxic payloads to tumors with radiolabeled molecules that recognize tumor-associated antigens. We have recently demonstrated the potential of an FDA approved, humanized anti-GD2 antibody, hu3F8, as a targeted delivery vector for radiopharmaceutical imaging of OS. The current study aims to advance this system for alpha-particle therapy of OS. METHODS: The hu3F8 antibody was radiolabeled with actinium-225, and the safety and therapeutic efficacy of the [225Ac]Ac-DOTA-hu3F8 were evaluated in both orthotopic murine xenografts of OS and spontaneously occurring OS in canines. RESULTS: Significant antitumor activity was proven in both cases, leading to improved overall survival. In the murine xenograft's case, tumor growth was delayed by 16-18 days compared to the untreated cohort as demonstrated by bioluminescence imaging. The results were further validated with magnetic resonance imaging at 33 days after treatment, and microcomputed tomography and planar microradiography post-mortem. Histological evaluations revealed radiation-induced renal toxicity, manifested as epithelial cell karyomegaly and suggestive polyploidy in the kidneys, suggesting rapid recovery of renal function after radiation damage. Treatment of the two canine patients delayed the progression of metastatic spread, with an overall survival time of 211 and 437 days and survival beyond documented metastasis of 111 and 84 days, respectively. CONCLUSION: This study highlights the potential of hu3F8-based alpha-particle therapy as a promising treatment strategy for OS.

Gel-forming therapeutic peptide exhibits sustained delivery and efficacy in a mouse model of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a particularly aggressive and invasive subtype of breast cancer that represents a major cause of death of women worldwide. Here we describe the efficacy of an integrin-binding antiangiogenic peptide in a variety of delivery methods and dosing conditions. This peptide, AXT201, demonstrated consistent anti-tumor efficacy when administered intraperitoneally, subcutaneously, and intratumorally, and retained this activity even when dosing frequency was reduced to once every two weeks. Finally, in vivo imaging and biodistribution studies of AXT201 showed a long-term persistence of at least 10 days at the site of injection and a stable detectable signal in the blood over 48 h, indicating a sustained release profile. Taken together, these findings indicate AXT201 exhibits favorable pharmacokinetic properties for a 20-mer peptide.

Early Normal Tissue Effects and Bone Marrow Relative Biological Effectiveness for an Actinium 225-Labeled HER2/neu-Targeting Antibody.

PURPOSE: In this study we determined the dose-independent relative biological effectiveness (RBE2) of bone marrow for an anti-HER2/neu antibody labeled with the alpha-particle emitter actinium 225 (225Ac). Hematologic toxicity is often a consequence of radiopharmaceutical therapy (RPT) administration, and dosimetric guidance to the bone marrow is required to limit toxicity. METHODS AND MATERIALS: Female neu/N transgenic mice (MMTV-neu) were intravenously injected with 0 to 16.65 kBq of the alpha-particle emitter labeled antibody, 225Ac-DOTA-7.16.4, and euthanized at 1 to 9 days after treatment. Complete blood counts were performed. Femurs and tibias were collected, and bone marrow was isolated from 1 femur and tibia and counted for radioactivity. Contralateral intact femurs were fixed, decalcified, and assessed by histology. Marrow cellularity was the biologic endpoint selected for RBE2 determination. For the reference radiation, both femurs of the mice were photon irradiated with 0 to 5 Gy using a small animal radiation research platform. RESULTS: Response as measured by cellularity for the alpha-particle emitter RPT (αRPT) RPT and the external beam radiation therapy were linear and linear quadratic, respectively, as a function of absorbed dose. The resulting dose-independent RBE2 for bone marrow was 6. CONCLUSIONS: As αRPT gains prominence, preclinical studies evaluating RBE in vivo will be important in relating to human experience with beta-particle emitter RPT. Such normal tissue RBE evaluations will help mitigate unexpected toxicity in αRPT.

Large-scale in vitro microdosimetry via live cell microscopy imaging: implications for radiosensitivity and RBE evaluations in alpha-emitter radiopharmaceutical therapy.

BACKGROUND: Alpha-emitter radiopharmaceutical therapy (αRPT) has shown promising outcomes in metastatic disease. However, the short range of the alpha particles necessitates dosimetry on a near-cellular spatial scale. Current knowledge on cellular dosimetry is primarily based on in vitro experiments using cell monolayers. The goal of such experiments is to establish cell sensitivity to absorbed dose (AD). However, AD cannot be measured directly and needs to be modeled. Current models, often idealize cells as spheroids in a regular grid (geometric model), simplify binding kinetics and ignore the stochastic nature of radioactive decay. It is unclear what the impact of such simplifications is, but oversimplification results in inaccurate and non-generalizable results, which hampers the rigorous study of the underlying radiobiology. METHODS: We systematically mapped out 3D cell geometries, clustering behavior, agent binding, internalization, and subcellular trafficking kinetics for a large cohort of live cells under representative experimental conditions using confocal microscopy. This allowed for realistic Monte Carlo-based (micro)dosimetry. Experimentally established surviving fractions of the HER2 + breast cancer cell line treated with a 212Pb-labelled anti-HER2 conjugate or external beam radiotherapy, anchored a rigorous statistical approach to cell sensitivity and relative biological effectiveness (RBE) estimation. All outcomes were compared to a reference geometric model, which allowed us to determine which aspects are crucial model components for the proper study of the underlying radiobiology. RESULTS: In total, 567 cells were measured up to 26 h post-incubation. Realistic cell clustering had a large (2x), and cell geometry a small (16.4% difference) impact on AD, compared to the geometric model. Microdosimetry revealed that more than half of the cells do not receive any dose for most of the tested conditions, greatly impacting cell sensitivity estimates. Including these stochastic effects in the model, resulted in significantly more accurate predictions of surviving fraction and RBE (permutation test; p < .01). CONCLUSIONS: This comprehensive integration of the biological and physical aspects resulted in a more accurate method of cell survival modelling in αRPT experiments. Specifically, including realistic stochastic radiation effects and cell clustering behavior is crucial to obtaining generalizable radiobiological parameters.

Bone Marrow Relative Biological Effectiveness for a 212Pb-labeled Anti-HER2/neu Antibody.

PURPOSE: We have determined the in vivo relative biological effectiveness (RBE) of an alpha-particle-emitting radiopharmaceutical therapeutic agent (212Pb-labeled anti-HER2/neu antibody) for the bone marrow, a potentially dose-limiting normal tissue. METHODS AND MATERIALS: The RBE was measured in mice using femur marrow cellularity as the biological endpoint. External beam radiation therapy (EBRT), delivered by a small-animal radiation research platform was used as the reference radiation. Alpha-particle emissions were delivered by 212Bi after the decay of its parent nuclide 212Pb, which was conjugated onto an anti-HER2/neu antibody. The alpha-particle absorbed dose to the marrow after an intravenous administration (tail vein) of 122.1 to 921.3 kBq 212Pb-TCMC-7.16.4 was calculated. The mice were sacrificed at 0 to 7 days after treatment and the radioactivity from the femur bone marrow was measured. Changes in marrow cellularity were assessed by histopathology. RESULTS: The dose response for EBRT and 212Pb-anti-HER2/neu antibody were linear-quadratic and linear, respectively. On transforming the EBRT dose-response relationship into a linear relationship using the equivalent dose in 2-Gy fractions of external beam radiation formalism, we obtained an RBE (denoted RBE2) of 6.4, which is independent of cellularity and absorbed dose. CONCLUSIONS: Because hematologic toxicity is dose limiting in almost all antibody-based RPT, in vivo measurements of RBE are important in helping identify an initial administered activity in phase 1 escalation trials. Applying the RBE2 and assuming typical antibody clearance kinetics (biological half-life of 48 hours), using a modified blood-based dosimetry method, an average administered activity of approximately 185.5 MBq (5.0 mCi) per patient could be administered before hematologic toxicity is anticipated.

Anti-GD2 antibody for radiopharmaceutical imaging of osteosarcoma.

PURPOSE: Osteosarcoma (OS) is the most frequently diagnosed bone cancer in children with little improvement in overall survival in the past decades. The high surface expression of disialoganglioside GD2 on OS tumors and restricted expression in normal tissues makes it an ideal target for anti-OS radiopharmaceuticals. Since human and canine OS share many biological and molecular features, spontaneously occurring OS in canines has been an ideal model for testing new imaging and treatment modalities for human translation. In this study, we evaluated a humanized anti-GD2 antibody, hu3F8, as a potential delivery vector for targeted radiopharmaceutical imaging of human and canine OS. METHODS: The cross-reactivity of hu3F8 with human and canine OS cells and tumors was examined by immunohistochemistry and flow cytometry. The hu3F8 was radiolabeled with indium-111, and the biodistribution of [111In]In-hu3F8 was assessed in tumor xenograft-bearing mice. The targeting ability of [111In]In-hu3F8 to metastatic OS was tested in spontaneous OS canines. RESULTS: The hu3F8 cross reacts with human and canine OS cells and canine OS tumors with high binding affinity. Biodistribution studies revealed selective uptake of [111In]In-hu3F8 in tumor tissue. SPECT/CT imaging of spontaneous OS canines demonstrated avid uptake of [111In]In-hu3F8 in all metastatic lesions. Immunohistochemistry confirmed the extensive binding of radiolabeled hu3F8 within both osseous and soft lesions. CONCLUSION: This study demonstrates the feasibility of targeting GD2 on OS cells and spontaneous OS canine tumors using hu3F8-based radiopharmaceutical imaging. Its ability to deliver an imaging payload in a targeted manner supports the utility of hu3F8 for precision imaging of OS and potential future use in radiopharmaceutical therapy.

212Pb-conjugated anti-rat HER2/neu antibody against a neu-N derived murine mammary carcinoma cell line: cell kill and RBE in vitro.

PURPOSE: In the current work, the RBE of a 212Pb-conjugated anti-HER2/neu antibody construct has been evaluated, in vitro, by colony formation assay. The RBE was estimated by comparing two absorbed dose-survival curves: the first obtained from the conjugated 212Pb experiments (test radiation), the second obtained by parallel experiments of single bolus irradiation of external beam (reference radiation). MATERIALS AND METHODS: Mammary carcinoma NT2.5 cells were treated with (0-3.70) kBq/ml of radiolabeled antibody. Nonspecific binding was assessed with addition of excess amount of unlabeled antibody. The colony formation curves were converted from activity concentration to cell nucleus absorbed dose by simulating the decay and transport of all daughter and secondary particles of 212Pb, using the Monte Carlo code GEANT 4. RESULTS: The radiolabeled antibody yielded an RBE of 8.3 at 37% survival and a survival independent RBE (i.e. RBE2) of 9.9. Unbound/untargeted 212Pb-labeled antibody, as obtained in blocking experiments yielded minimal alpha-particle radiation to cells. Conclusions: These results further highlight the importance of specific targeting toward achieving tumor cell kill and low toxicity to normal tissue.

Cu(II) adsorption on 2-thiouracil-modified Luffa cylindrica biochar fibres from artificial and real samples, and competition reactions with U(VI).

The adsorption of Cu(II) ions by biochar fibres prior and after modification with 2-thiouracil on real and artificial samples has been studied by batch-type adsorption experiments, FTIR and XPS spectroscopy and competition reactions using U(VI) ions as competitor cations. The experimental data of the artificial samples clearly show that the modified material presents extraordinary higher affinity for Cu(II) ions even in the acidic pH range, the spectroscopic data indicate the formation of inner-sphere complexes and the competition reactions significantly higher selectivity of the 2-thiouracil modified biochar fibres for Cu(II). The 2-thiouracil-modified biochar fibres have been successfully applied to acid mine drainage (AMD) samples regarding the selective separation of Cu(II) ions from "real" samples. Regarding the desorption of copper from the biochar surface, although 100% copper recovery was achieved by eluting the metal ion using 1 M HNO3, the deterioration of the modified biochar fibers due to extensive 2-thiouracil release from the biochar surface limits the applicability of the present adsorbent in routine and large-scale applications.