IgA antibody immunotherapy targeting GD2 is effective in preclinical neuroblastoma models.

BACKGROUND: Immunotherapy targeting GD2 is very effective against high-risk neuroblastoma, though administration of anti-GD2 antibodies induces severe and dose-limiting neuropathic pain by binding GD2-expressing sensory neurons. Previously, the IgG1 ch14.18 (dinutuximab) antibody was reformatted into the IgA1 isotype, which abolishes neuropathic pain and induces efficient neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) via activation of the Fc alpha receptor (FcαRI/CD89). METHODS: To generate an antibody suitable for clinical application, we engineered an IgA molecule (named IgA3.0 ch14.18) with increased stability, mutated glycosylation sites and substituted free (reactive) cysteines. The following mutations were introduced: N45.2G and P124R (CH1 domain), C92S, N120T, I121L and T122S (CH2 domain) and a deletion of the tail piece P131-Y148 (CH3 domain). IgA3.0 ch14.18 was evaluated in binding assays and in ADCC and antibody-dependent cellular phagocytosis (ADCP) assays with human, neuroblastoma patient and non-human primate effector cells. We performed mass spectrometry analysis of N-glycans and evaluated the impact of altered glycosylation in IgA3.0 ch14.18 on antibody half-life by performing pharmacokinetic (PK) studies in mice injected intravenously with 5 mg/kg antibody solution. A dose escalation study was performed to determine in vivo efficacy of IgA3.0 ch14.18 in an intraperitoneal mouse model using 9464D-GD2 neuroblastoma cells as well as in a subcutaneous human xenograft model using IMR32 neuroblastoma cells. Binding assays and PK studies were compared with one-way analysis of variance (ANOVA), ADCC and ADCP assays and in vivo tumor outgrowth with two-way ANOVA followed by Tukey's post-hoc test. RESULTS: ADCC and ADCP assays showed that particularly neutrophils and macrophages from healthy donors, non-human primates and patients with neuroblastoma are able to kill neuroblastoma tumor cells efficiently with IgA3.0 ch14.18. IgA3.0 ch14.18 contains a more favorable glycosylation pattern, corresponding to an increased antibody half-life in mice compared with IgA1 and IgA2. Furthermore, IgA3.0 ch14.18 penetrates neuroblastoma tumors in vivo and halts tumor outgrowth in both 9464D-GD2 and IMR32 long-term tumor models. CONCLUSIONS: IgA3.0 ch14.18 is a promising new therapy for neuroblastoma, showing (1) increased half-life compared to natural IgA antibodies, (2) increased protein stability enabling effortless production and purification, (3) potent CD89-mediated tumor killing in vitro by healthy subjects and patients with neuroblastoma and (4) antitumor efficacy in long-term mouse neuroblastoma models.

[212Pb]Pb-eSOMA-01: A Promising Radioligand for Targeted Alpha Therapy of Neuroendocrine Tumors.

Peptide receptor radionuclide therapy (PRRT) has been applied to the treatment of neuroendocrine tumors (NETs) for over two decades. However, improvement is still needed, and targeted alpha therapy (TAT) with alpha emitters such as lead-212 (212Pb) represents a promising avenue. A series of ligands based on octreotate was developed. Lead-203 was used as an imaging surrogate for the selection of the best candidate for the studies with lead-212. 203/212Pb radiolabeling and in vitro assays were carried out, followed by SPECT/CT imaging and ex vivo biodistribution in NCI-H69 tumor-bearing mice. High radiochemical yields (≥99%) and purity (≥96%) were obtained for all ligands. [203Pb]Pb-eSOMA-01 and [203Pb]Pb-eSOMA-02 showed high stability in PBS and mouse serum up to 24 h, whereas [203Pb]Pb-eSOMA-03 was unstable in those conditions. All compounds exhibited a nanomolar affinity (2.5-3.1 nM) for SSTR2. SPECT/CT images revealed high tumor uptake at 1, 4, and 24 h post-injection of [203Pb]Pb-eSOMA-01/02. Ex vivo biodistribution studies confirmed that the highest uptake in tumors was observed with [212Pb]Pb-eSOMA-01. [212Pb]Pb-eESOMA-01 displayed the highest absorbed dose in the tumor (35.49 Gy/MBq) and the lowest absorbed dose in the kidneys (121.73 Gy/MBq) among the three tested radioligands. [212Pb]Pb-eSOMA-01 is a promising candidate for targeted alpha therapy of NETs. Further investigations are required to confirm its potential.

Evaluating the Targeting of a Staphylococcus-aureus-Infected Implant with a Radiolabeled Antibody In Vivo.

Implant infections caused by Staphylococcus aureus are difficult to treat due to biofilm formation, which complicates surgical and antibiotic treatment. We introduce an alternative approach using monoclonal antibodies (mAbs) targeting S. aureus and provide evidence of the specificity and biodistribution of S.-aureus-targeting antibodies in a mouse implant infection model. The monoclonal antibody 4497-IgG1 targeting wall teichoic acid in S. aureus was labeled with indium-111 using CHX-A"-DTPA as a chelator. Single Photon Emission Computed Tomography/computed tomographyscans were performed at 24, 72 and 120 h after administration of the 111In-4497 mAb in Balb/cAnNCrl mice with a subcutaneous implant that was pre-colonized with S. aureus biofilm. The biodistribution of this labelled antibody over various organs was visualized and quantified using SPECT/CT imaging, and was compared to the uptake at the target tissue with the implanted infection. Uptake of the 111In-4497 mAbs at the infected implant gradually increased from 8.34 %ID/cm3 at 24 h to 9.22 %ID/cm3 at 120 h. Uptake at the heart/blood pool decreased over time from 11.60 to 7.58 %ID/cm3, whereas the uptake in the other organs decreased from 7.26 to less than 4.66 %ID/cm3 at 120 h. The effective half-life of 111In-4497 mAbs was determined to be 59 h. In conclusion, 111In-4497 mAbs were found to specifically detect S. aureus and its biofilm with excellent and prolonged accumulation at the site of the colonized implant. Therefore, it has the potential to serve as a drug delivery system for the diagnostic and bactericidal treatment of biofilm.

Synthesis and Evaluation of Two Long-Acting SSTR2 Antagonists for Radionuclide Therapy of Neuroendocrine Tumors.

Somatostatin receptor subtype 2 (SSTR2) has become an essential target for radionuclide therapy of neuroendocrine tumors (NETs). JR11 was introduced as a promising antagonist peptide to target SSTR2. However, due to its rapid blood clearance, a better pharmacokinetic profile is necessary for more effective treatment. Therefore, two JR11 analogs (8a and 8b), each carrying an albumin binding domain, were designed to prolong the blood residence time of JR11. Both compounds were labeled with lutetium-177 and evaluated via in vitro assays, followed by in vivo SPECT/CT imaging and ex vivo biodistribution studies. [177Lu]Lu-8a and [177Lu]Lu-8b were obtained with high radiochemical purity (>97%) and demonstrated excellent stability in PBS and mouse serum (>95%). [177Lu]Lu-8a showed better affinity towards human albumin compared to [177Lu]Lu-8b. Further, 8a and 8b exhibited binding affinities 30- and 48-fold lower, respectively, than that of the parent peptide JR11, along with high cell uptake and low internalization rate. SPECT/CT imaging verified high tumor accumulation for [177Lu]Lu-8a and [177Lu]Lu-JR11 at 4, 24, 48, and 72 h post-injection, but no tumor uptake was observed for [177Lu]Lu-8b. Ex vivo biodistribution studies revealed high and increasing tumor uptake for [177Lu]Lu-8a. However, its extended blood circulation led to an unfavorable biodistribution profile for radionuclide therapy.

Cancer-Associated Fibroblasts as Players in Cancer Development and Progression and Their Role in Targeted Radionuclide Imaging and Therapy.

Cancer Associated Fibroblasts (CAFs) form a major component of the tumour microenvironment, they have a complex origin and execute diverse functions in tumour development and progression. As such, CAFs constitute an attractive target for novel therapeutic interventions that will aid both diagnosis and treatment of various cancers. There are, however, a few limitations in reaching successful translation of CAF targeted interventions from bench to bedside. Several approaches targeting CAFs have been investigated so far and a few CAF-targeting tracers have successfully been developed and applied. This includes tracers targeting Fibroblast Activation Protein (FAP) on CAFs. A number of FAP-targeting tracers have shown great promise in the clinic. In this review, we summarize our current knowledge of the functional heterogeneity and biology of CAFs in cancer. Moreover, we highlight the latest developments towards theranostic applications that will help tumour characterization, radioligand therapy and staging in cancers with a distinct CAF population.

EXIRAD-3D: Fast automated three-dimensional autoradiography.

INTRODUCTION: Autoradiography is an established technique for high-resolution imaging of radiolabelled molecules in biological tissue slices. Unfortunately, creating a 3D image from a set of these 2D images is extremely time-consuming and error-prone. MicroSPECT systems provide such 3D images but have a low resolution. Here we present EXIRAD-3D, a fast automated method as an alternative for 3D autoradiography from coupes based on ultra-high resolution microSPECT technology. METHODS: EXIRAD-3D uses a very small bore focusing multi-pinhole collimator mounted in a SPECT system with stationary detectors (U-SPECT/CT, MILabs B.V. The Netherlands) using a sample holder with integrated tissue cooling to avoid activity leaking or tissue deformation during the scan. The system performance was experimentally evaluated using various phantoms and tissue samples of animals in vivo injected with technetium-99m and iodine-123. RESULTS: The reconstructed spatial resolution obtained with a Derenzo hot rod phantom was 120 µm (or 1.7 nl). The voxel values of a syringe phantom image appear to be uniform and scale linearly with activity. Uptake in tiny details of the mouse knee joint, thyroid, and kidney could be clearly visualized. CONCLUSION: EXIRAD-3D opens up the possibility for fast and quantitative 3D imaging of radiolabelled molecules at a resolution far better than in vivo microSPECT and saves tremendous amounts of work compared to obtaining 3D data from a set of 2D autoradiographs. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: EXIRAD-3D offers superior image resolution over microSPECT, and it can be a very efficient alternative for autoradiography in pharmaceutical and biological studies.

Radiolabeled cCPE Peptides for SPECT Imaging of Claudin-4 Overexpression in Pancreatic Cancer.

Overexpression of tight-junction protein claudin-4 has been detected in primary and metastatic pancreatic cancer tissue and is associated with better prognosis in patients. Noninvasive measurement of claudin-4 expression by imaging methods could provide a means for accelerating detection and stratifying patients into risk groups. Clostridium perfringens enterotoxin (CPE) is a natural ligand for claudin-4 and holds potential as a targeting vector for molecular imaging of claudin-4 overexpression. A glutathione S-transferases (GST)-tagged version of the C terminus of CPE (cCPE) was previously used to delineate claudin-4 overexpression by SPECT but showed modest binding affinity and slow blood clearance in vivo. Methods: On the basis of the crystal structure of cCPE, a series of smaller cCPE194-319 mutants with putatively improved binding affinity for claudin-4 was generated by site-directed mutagenesis. All peptides were conjugated site-specifically on a C-terminal cysteine using maleimide-diethylenetriamine pentaacetate to enable radiolabeling with 111In. The binding affinity of all radioconjugates was evaluated in claudin-4-expressing PSN-1 cells and HT1080-negative controls. The specificity of all cCPE mutants to claudin-4 was assessed in HT1080 cells stably transfected with claudin-4. SPECT/CT imaging of BALB/c nude mice bearing PSN-1 or HT1080 tumor xenografts was performed to determine the claudin-4-targeting ability of these peptides in vivo. Results: Uptake of all cCPE-based radioconjugates was significantly higher in PSN-1 cells than in HT1080-negative controls. All peptides showed a marked improvement in affinity for claudin-4 in vitro when compared with previously reported values (dissociation constant: 2.2 ± 0.8, 3 ± 0.1, 4.2 ± 0.5, 10 ± 0.9, and 9.7 ± 0.7 nM). Blood clearance of [111In]In-cCPE194-319, as measured by SPECT, was considerably faster than that of [111In]In-cCPE.GST (half-life, <1 min). All radiopeptides showed significantly higher accumulation in PSN-1 xenografts than in HT1080 tumors at 90 min after injection of the tracer ([111In]In-cCPE194-319, 2.7 ± 0.8 vs. 0.4 ± 0.1 percentage injected dose per gram [%ID/g], P < 0.001; [111In]In-S313A, 2.3 ± 0.9 vs. 0.5 ± 0.1 %ID/g, P < 0.01; [111In]In-S307A + N309A + S313A, 2 ± 0.4 vs. 0.3 ± 0.1 %ID/g, P < 0.01; [111In]In-D284A, 2 ± 0.2 vs. 0.7 ± 0.1 %ID/g, P < 0.05; [111In]In-L254F + K257D, 6.3 ± 0.9 vs. 0.7 ± 0.2 %ID/g, P < 0.001). Conclusion: These optimized cCPE-based SPECT imaging agents show great promise as claudin-4-targeting vectors for in vivo imaging of claudin-4 overexpression in pancreatic cancer.

Imaging of Claudin-4 in Pancreatic Ductal Adenocarcinoma Using a Radiolabelled Anti-Claudin-4 Monoclonal Antibody.

PURPOSE: Despite its widespread use, the positron emission tomography (PET) radiotracer 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has been shown in clinical settings to be ineffective for improving early diagnosis of pancreatic ductal adenocarcinoma (PDAC). A promising biomarker for PDAC detection is the tight junction protein claudin-4. The purpose of this study was to evaluate a new single-photon emission computed tomography (SPECT) imaging agent, [111In]anti-claudin-4 mAb, with regard to its ability to allow visualisation of claudin-4 in a xenograft and a genetically engineered mouse model of PDAC. PROCEDURES: The ability of [111In]anti-claudin-4 mAb to selectively target claudin-4 was assessed using two human xenograft tumour models with differential claudin-4 status in mice. [111In]anti-claudin-4 mAb was also used to detect PDAC development in genetically engineered KPC mice. The PDAC status of these mice was confirmed with [18F]FDG-PET, magnetic resonance imaging (MRI), histology, and immunofluorescence microscopy. RESULTS: High uptake of [111In]anti-claudin-4 mAb was observed in PDAC xenografts in mice, reaching 16.9 ± 4.5 % of injected dose per gram (% ID/g) at 72 h post-injection. This uptake was mediated specifically by the expression of claudin-4. Uptake of [111In]anti-claudin-4 mAb also enabled clear visualisation of spontaneous PDAC formation in KPC mice. CONCLUSIONS: [111In]anti-claudin-4 mAb allows non-invasive detection of claudin-4 upregulation during development of PDAC and could potentially be used to aid in the early detection and characterisation of this malignancy.

Development of a T-cell Receptor Mimic Antibody against Wild-Type p53 for Cancer Immunotherapy.

The tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Because of its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anticancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of MHC I and can be bound by T-cell receptors (TCR). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201-presented wild-type p53 T-cell epitope, p5365-73(RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitroIn vivo, the antibody targets p5365-73 peptide-expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy. Cancer Res; 77(10); 2699-711. ©2017 AACR.

Imaging the DNA damage response with PET and SPECT.

DNA integrity is constantly challenged by endogenous and exogenous factors that can alter the DNA sequence, leading to mutagenesis, aberrant transcriptional activity, and cytotoxicity. Left unrepaired, damaged DNA can ultimately lead to the development of cancer. To overcome this threat, a series of complex mechanisms collectively known as the DNA damage response (DDR) are able to detect the various types of DNA damage that can occur and stimulate the appropriate repair process. Each DNA damage repair pathway leads to the recruitment, upregulation, or activation of specific proteins within the nucleus, which, in some cases, can represent attractive targets for molecular imaging. Given the well-established involvement of DDR during tumorigenesis and cancer therapy, the ability to monitor these repair processes non-invasively using nuclear imaging techniques may facilitate the earlier detection of cancer and may also assist in monitoring response to DNA damaging treatment. This review article aims to provide an overview of recent efforts to develop PET and SPECT radiotracers for imaging of DNA damage repair proteins.