[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.</p

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

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. Due to its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anti-cancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of major histocompatibility class I (MHC I), and can be bound by T cell receptors (TCRs). 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 vitro. In 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

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

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

Imaging p53 in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) represents 90% of pancreatic cancer cases and is characterised by poor survival rates and resistance to current therapeutic regimes as the majority of patients present with an already advanced disease at the time of diagnosis. Mutations of the TP53 tumour suppressor gene are a frequent event in tumourigenesis and appear in around 75% of pancreatic cancer cases just before the full development of PDAC and metastasis into the surrounding tissues. Molecular imaging tools, such as SPECT, using monoclonal antibodies specific towards tumour associated antigens, could aid the in vivo characterisation of biological processes and provide a non-invasive method for early diagnosis of cancer. Toward this end, a commercially available mouse monoclonal antibody against total p53 protein (anti-p53 (1C12)) was selected. Anti-p53 (1C12) was first evaluated in vitro regarding its specificity and affinity in cell lines with variable p53 status, derived from a genetically engineered mouse model of PDAC (KPC mice). The subcellular localisation of the p53 protein in the cell lines used was also studied. Following the selection of the antibody, the anti-p53 (1C12) was conjugated to the cell penetrating peptide TAT to facilitate cellular and nuclear translocation and then to p-SCN-Bn-DTPA to allow labeling with 111In. Further in vitro evaluation was performed on the conjugated antibody, conferring subcellular translocation in fixed cells, unaffected binding affinity, favourable radiochemical yield and purity, and stability of the radioconjugate in serum. Retention of the radioconjugates was also observed and was signicantly different when compared to radiolabeled non-specific IgG1-TAT in cells harbouring mutant p53. The ability of 111In-BnDTPA-p53 (1C12)-TAT to selectively target endogenously expressed p53 was assessed in vivo using mouse allograft tumour models of the cell lines evaluated in vitro, and in genetically engineered KPC mice. This provided a preliminary ''proof-of-principle" concept for diagnostic detection of PDAC. The knowledge acquired from the current study may be used to develop a first imaging tracer against p53 which could significantly improve the biological evaluation of cancer.</p

Imaging p53 in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) represents 90% of pancreatic cancer cases and is characterised by poor survival rates and resistance to current therapeutic regimes as the majority of patients present with an already advanced disease at the time of diagnosis. Mutations of the TP53 tumour suppressor gene are a frequent event in tumourigenesis and appear in around 75% of pancreatic cancer cases just before the full development of PDAC and metastasis into the surrounding tissues. Molecular imaging tools, such as SPECT, using monoclonal antibodies specific towards tumour associated antigens, could aid the in vivo characterisation of biological processes and provide a non-invasive method for early diagnosis of cancer. Toward this end, a commercially available mouse monoclonal antibody against total p53 protein (anti-p53 (1C12)) was selected. Anti-p53 (1C12) was first evaluated in vitro regarding its specificity and affinity in cell lines with variable p53 status, derived from a genetically engineered mouse model of PDAC (KPC mice). The subcellular localisation of the p53 protein in the cell lines used was also studied. Following the selection of the antibody, the anti-p53 (1C12) was conjugated to the cell penetrating peptide TAT to facilitate cellular and nuclear translocation and then to p-SCN-Bn-DTPA to allow labeling with 111In. Further in vitro evaluation was performed on the conjugated antibody, conferring subcellular translocation in fixed cells, unaffected binding affinity, favourable radiochemical yield and purity, and stability of the radioconjugate in serum. Retention of the radioconjugates was also observed and was signicantly different when compared to radiolabeled non-specific IgG1-TAT in cells harbouring mutant p53. The ability of 111In-BnDTPA-p53 (1C12)-TAT to selectively target endogenously expressed p53 was assessed in vivo using mouse allograft tumour models of the cell lines evaluated in vitro, and in genetically engineered KPC mice. This provided a preliminary ''proof-of-principle" concept for diagnostic detection of PDAC. The knowledge acquired from the current study may be used to develop a first imaging tracer against p53 which could significantly improve the biological evaluation of cancer.</p

Development of 203Pb-labeled SSTR2 radioligands for neuroendocrine tumors (NETs) imaging

Objectives : Development of theranostic pairs is generally guided by the biodistribution and pharmacokinetic properties of the imaging agent. Indeed, conducting such studies with beta or alpha emitters, such as lead-212, can be challenging. Therefore, it is often preferable to use a surrogate to facilitate detection of radioactivity in tissues, follow distribution of the radioactive drug over time by non-invasive imaging, and ease radiation safety. In this context, lead203, a gamma emitter (t1/2 =52 h), is perfectly adapted to study the distribution of 212Pb-labeled drugs, as it provides chemically identical companion diagnostics. Our objective was therefore to design and pre-clinically evaluate two novel 203Pb-labeled SSTR2 ligands for theranostic application of NETs. Methods : Two octreotate derivatives, containing a 4-(aminomethyl)cyclohexane-1-carbonyl (Amcha) or a 4-amino-1-carboxymethyl-piperidinyl (Pip) linker, were synthesized by SPPS. In vitro competitive binding assays with [111In]In-DOTATATE were performed using CHO-K1 cell membranes over-expressing human SSTR2. Both ligands were labeled with 203Pb in sodium acetate buffer for 20 minutes at room temperature and analyzed by iTLC and radioHPLC. LogD7.4 and stability in serum were also determined. SPECT/CT imaging was performed at 1, 4 and 24 h post-injection in H69 tumorbearing mice, followed by ex-vivo biodistribution. Results : DO3AM-Amcha-TATE and DO3AM-Pip-TATE were successfully synthesized with a yield and purity of 10% and 99%, respectively. Both ligands showed low nanomolar binding affinity for SSTR2 and hydrophilic behavior. DO3AM-Amcha-TATE and DO3AM-PipTATE were labeled with 203Pb with a radiochemical yield and purity exceeding 98%. Stability studies revealed that our 203Pb-labeled octreotate derivatives were stable in serum for 24 h. Image analysis of the two radioligands showed high uptake at 1 h in the SSTR2-positive tumors (4.0 ± 0.57 and 3.3 ± 1.08%ID/mL for [203Pb]Pb-DO3AMAmcha-TATE and [203Pb]Pb-DO3AM-PIP-TATE, respectively), as well as in the kidneys ([203Pb]Pb-DO3AM-Amcha-TATE: 10.6 ± 1.7%ID/mL and [203Pb]Pb-DO3AM-Pip-TATE: 12.8 ± 1.80%ID/mL), but also at 24 h p.i. (tumors: 2.4 ± 0.27%ID/mL and 2.2 ± 0.64%ID/mL; kidneys: 3.6 ± 1.2%ID/mL and 6.6 ± 1.17%ID/mL for [203Pb]Pb-DO3AM-Amcha-TATE and [203Pb]Pb-DO3AM-Pip-TATE, respectively). The tumor-to-kidney ratio increased over time for [203Pb]Pb-DO3AM-Amcha-TATE (0.4 at 1 h to 0.7 at 24 h p.i.), while it remained constant for the [203Pb] Pb-DO3AM-Pip-TATE (0.26 at 1 h to 0.34 at 24 h p.i.). Ex-vivo biodistribution confirmed that administration of [203Pb]Pb-DO3AMPip-TATE resulted in a higher uptake in the pancreas and bones than [203Pb]Pb-DO3AM-Amcha-TATE. Conclusions : Our results indicated that these two ligands are promising candidates for imaging of SSTR2-positive tumors. Further investigations will be conducted with the 212Pb-labeled analogs to determine their potential as theranostic agents