Survival impact of [225Ac]Ac-DOTATOC alpha-therapy in a preclinical model of pancreatic neuroendocrine tumor liver micrometastases.

Although peptide radionuclide therapy (PRRT) using a somatostatin analog (SSA) radiolabeled with a beta- emitter: [177Lu]Lu-DOTATATE has shown a good clinical efficacy in neuroendocrine tumors (NETs), most of the patients only achieved tumoral stabilization and rare but severe long-term hematological toxicities have been reported. One of the promising options to improve PRRT is targeted alpha therapy. It is therefore essential to propose animal models that can mimic systemic spread disease, especially microscopic disease such as early stage of NET liver metastases to explore targeted alpha therapy. Herein, we report the evaluation of efficacy and toxicity of [225Ac]Ac-DOTATOC in an original preclinical murine model simulating the development of well-characterized liver metastases of pancreatic NETs with SSTR overexpression. METHODS: A mouse model of liver metastases of pancreatic NETs was developed by intraportal injection of AR42J cells and explored using [68 Ga]Ga-DOTATOC and [18F]F-FDG PET/MRI. Biodistribution study and radiation dosimetry of [225Ac]Ac-DOTATOC were determined in subcutaneous tumor-bearing NMRI-nude mice. Efficacy and toxicity were determined by intravenous injection of increasing activities of [225Ac]Ac-DOTATOC 10 days after intraportal graft. RESULTS: Liver tumors showed a high uptake of [68 Ga]Ga-DOTATOC and no uptake of [18F]F-FDG confirming the well-differentiated phenotype. All groups treated with [225Ac]Ac-DOTATOC showed a significant increase in overall survival compared with DOTATOC-treated mice, especially those treated with the highest activities: 53 days with 240 kBq (p = 0.0001), and 58 days with 2 × 120 kBq (p < 0.0001) vs 28 days with non-radiolabeled DOTATOC. On blood tests, a transient and moderate decreased in white blood cells count after treatment and no severe hepatic or renal toxicity were observed after treatment which was consistent with pathological and radiation dosimetry findings. CONCLUSION: [225Ac]Ac-DOTATOC exhibit a favorable efficacy and toxicity profile in a mouse model of liver micrometastatic pancreatic NET.

211 At and 125 I-Labeling of (Hetero)Aryliodonium Ylides: Astatine Wins Again.

Despite the growing interest in radioiodine and 211 At-labeled radiopharmaceuticals, the search for radiolabeling reactions has been somewhat neglected, resulting in a limited number of available radiosynthetic strategies. Herein we report a comparative study of nucleophilic 125 I and 211 At-labeling of aryliodonium ylides. Whereas radioiodination efficiency was low, 211 At-labeling performed efficiently on a broad scope of precursors. The most activated aryliodonium ylides led rapidly to quantitative reactions at room temperature in acetonitrile. For deactivated precursors, heating up to 90 °C in glyme and addition of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) as radical scavenger appeared essential to avoid precursor degradation and to achieve high radiochemical yields and molar activity. The approach was applied successfully to the preparation of 4-[211 At]astatophenylalanine (4-APA), an amino acid derivative increasingly studied as radiotherapeutic drug for cancers. This validated aryliodonium ylides as a valuable tool for nucleophilic 211 At-labeling and will complement the short but now growing list of available astatination reactions.

FLT3 ligand plasma levels have no impact on outcomes after allotransplant in acute leukemia.

OBJECTIVE: This study was designed to assess the impact on outcomes of early soluble Fms-like tyrosine kinase 3 ligand concentrations (sFLc) in patients receiving an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). METHODS: This was a prospective monocentric study including all allo-HSCT patients included in the previous FLAM/FLAL study (Peterlin et al., 2019). Blood samples collected before the start of conditioning then post-transplant were frozen, stored and tested by ELISA. The parameters considered were hematopoietic recoveries, Leukemia Free Survival and Overall Survival, acute and chronic GVHD, grade 3 or 4 acute and/or extensive chronic GVHD-free and relapse-free survival (GRFS). RESULTS: Forty-one patients were included, a total of 179 samples were assayed for sFLc. There was no impact of sFLc levels (<=median vs> median) on acute and chronic GVHD incidences, LFS, OS nor GRFS. CONCLUSION: At variance with induction results for AML (Peterlin et al., 2019) endogenous sFLc do not appear to be a prognostic marker at the time of or after allo-HSCT. Even though the results are negatives, this is, to the best of our knowledge, the only prospective series specifically addressing the question of sFLc impact after allo-HSCT in acute leukemias.

Tocilizumab in combination with a standard induction chemotherapy in acute myeloid leukaemia patients (TOCILAM study): a single-centre, single-arm, phase 1 trial.

Background: In acute myeloid leukaemia (AML), interleukin-6 (IL-6) promotes chemo-resistance and its levels correlate with poor prognosis. IL-6 blockade may represent a promising therapeutic strategy. We aimed to test, tocilizumab, an anti-IL-6 receptor (R) monoclonal antibody in combination with standard intensive AML induction chemotherapy. Methods: This investigator-initiated single-centre phase 1 trial was conducted at Nantes University Hospital in France. According to a continual reassessment method, three escalating doses were tested of intravenous (IV) tocilizumab (4, 6, and 8 mg/kg) administered at day (d) 8 of a standard AML induction chemotherapy (IV idarubicine 8 mg/m2 d1 to d5 + IV cytarabine 100 mg/m2 d1 to d7). All adults (aged ≥ 18 years) with an Eastern Cooperative Oncology Group performance status of 0-2 and with a newly diagnosed (excluding patients with a favourable risk according to ELN-2017 classification if <60 year-old) or a relapsed/refractory AML were eligible. The primary objective was to determine the maximum tolerated dose of tocilizumab to administrate with a standard intensive AML induction. Safety outcomes were continuously monitored for at each participant contact. This trial is registered with ClinicalTrials.gov, NCT04547062. Findings: Between Dec 29, 2020 and Dec 1, 2022, 12 patients were enrolled, of whom 75% had an ELN-2017 high-risk profile, and were treated with tocilizumab- two patients at 4 mg/kg, two at 6 mg/kg and eight at 8 mg/kg of tocilizumab. No dose-limiting toxicity related to tocilizumab was documented. There were nine serious adverse events, none of which were related to tocilizumab, and there was no treatment-related deaths. MTD was thus not reached. Two deaths occurred during induction. In the remaining ten evaluable patients, nine responded to treatment. Interpretation: The combination of tocilizumab with standard AML intensive induction appears to be safe and resulting responses are encouraging. A dose of 8 mg/kg of tocilizumab given at day 8 of induction could be used for further phase 2/3 studies. Funding: The Leucémie Espoir Atlantique Famille (LEAF)-"Tous avec Fabien" association.

Preclinical Evaluation of a 64Cu-Based Theranostic Approach in a Murine Model of Multiple Myeloma.

Although the concept of theranostics is neither new nor exclusive to nuclear medicine, it is a particularly promising approach for the future of nuclear oncology. This approach is based on the use of molecules targeting specific biomarkers in the tumour or its microenvironment, associated with optimal radionuclides which, depending on their emission properties, allow the combination of diagnosis by molecular imaging and targeted radionuclide therapy (TRT). Copper-64 has suitable decay properties (both ß+ and ß- decays) for PET imaging and potentially for TRT, making it both an imaging and therapy agent. We developed and evaluated a theranostic approach using a copper-64 radiolabelled anti-CD138 antibody, [64Cu]Cu-TE1PA-9E7.4 in a MOPC315.BM mouse model of multiple myeloma. PET imaging using [64Cu]Cu-TE1PA-9E7.4 allows for high-resolution PET images. Dosimetric estimation from ex vivo biodistribution data revealed acceptable delivered doses to healthy organs and tissues, and a very encouraging tumour absorbed dose for TRT applications. Therapeutic efficacy resulting in delayed tumour growth and increased survival without inducing major or irreversible toxicity has been observed with 2 doses of 35 MBq administered at a 2-week interval. Repeated injections of [64Cu]Cu-TE1PA-9E7.4 are safe and can be effective for TRT application in this syngeneic preclinical model of MM.

Targeted Alpha Particle Therapy Remodels the Tumor Microenvironment and Improves Efficacy of Immunotherapy.

PURPOSE: The tumor microenvironment (TME) can severely impair immunotherapy efficacy by repressing the immune system. In a multiple myeloma (MM) murine model, we investigated the impact of targeted alpha particle therapy (TAT) on the immune TME. TAT was combined with an adoptive cell transfer of CD8 T cells (ACT), and the mechanisms of action of this combination were assessed at the tumor site. METHODS AND MATERIALS: This combination treatment was conducted in a syngeneic MM murine model grafted subcutaneously. TAT was delivered by intravenous injection of a bismuth-213 radiolabeled anti-CD138 antibody. To strengthen antitumor immune response, TAT was combined with an ACT of tumor-specific CD8+ OT-1 T-cells. The tumors were collected and the immune TME analyzed by flow cytometry, immunohistochemistry, and ex vivo T-cell motility assay on tumor slices. The chemokine and cytokine productions were also assessed by quantitative reverse transcription polymerase chain reaction. RESULTS: Tumor-specific CD8+ OT-1 T cells infiltrated the tumors after ACT. However, only treatment with TAT resulted in regulatory CD4 T-cell drop and transient increased production of interleukin-2, CCL-5, and interferon-γ within the tumor. Moreover, OT-1 T-cell recruitment and motility were increased on tumor slices from TAT-treated mice, as observed via ex vivo time lapse, contributing to a more homogeneous distribution of OT-1 T cells in the tumor. Subsequently, the tumor cells increased PD-L1 expression, antitumor cytokine production decreased, and OT-1 T-cells overexpressed exhaustion markers, suggesting an exhaustion of the immune response. CONCLUSION: Combining TAT and ACT seems to transiently remodel the cold TME, improving ACT efficiency. The immune response then leads to the establishment of other tumor cell resistance mechanisms.

Anti-Tumor Efficacy of PD-L1 Targeted Alpha-Particle Therapy in a Human Melanoma Xenograft Model.

PD-L1 (programmed death-ligand 1, B7-H1, CD274), the ligand for PD-1 inhibitory receptor, is expressed on various tumors, and its expression is correlated with a poor prognosis in melanoma. Anti-PD-L1 mAbs have been developed along with anti-CTLA-4 and anti-PD-1 antibodies for immune checkpoint inhibitor (ICI) therapy, and anti-PD-1 mAbs are now used as first line treatment in melanoma. However, many patients do not respond to ICI therapies, and therefore new treatment alternatives should be developed. Because of its expression on the tumor cells and on immunosuppressive cells within the tumor microenvironment, PD-L1 represents an interesting target for targeted alpha-particle therapy (TAT). We developed a TAT approach in a human melanoma xenograft model that stably expresses PD-L1 using a 213Bi-anti-human-PD-L1 mAb. Unlike treatment with unlabeled anti-human-PD-L1 mAb, TAT targeting PD-L1 significantly delayed melanoma tumor growth and improved animal survival. A slight decrease in platelets was observed, but no toxicity on red blood cells, bone marrow, liver or kidney was induced. Anti-tumor efficacy was associated with specific tumor targeting since no therapeutic effect was observed in animals bearing PD-L1 negative melanoma tumors. This study demonstrates that anti-PD-L1 antibodies may be used efficiently for TAT treatment in melanoma.

A new cytokine-based dynamic stratification during induction is highly predictive of survivals in acute myeloid leukemia.

The aim of this study was to assess the potential impact of the kinetics of serum levels of seven cytokines during induction in acute myeloid leukemia (AML) patients. Indeed, the role of cytokines, in the pathophysiology and response to therapy of AML patients, remains under investigation. Here, we report on the impact of peripheral levels of two cytokines, the Fms-like tyrosine kinase 3 ligand (FL) and interleukin-6 (IL-6), evaluated during first-line intensive induction. A new risk stratification can be proposed, which supersedes the ELN 2017 classification to predict survivals in AML patients by examining the kinetic profile of these cytokines during the induction phase. It segregates three groups of, respectively, high-risk, characterized by a stagnation of low FL levels, intermediate risk, with dynamic increasing FL levels and high IL-6 at day 22, and favorable risk with increasing FL levels but low IL-6 at day 22.

Targeted-Alpha-Therapy Combining Astatine-211 and anti-CD138 Antibody in A Preclinical Syngeneic Mouse Model of Multiple Myeloma Minimal Residual Disease.

Despite therapeutic progress in recent years with the introduction of targeted therapies (daratumumab, elotuzumab), multiple myeloma remains an incurable cancer. The question is therefore to investigate the potential of targeted alpha therapy, combining an anti-CD138 antibody with astatine-211, to destroy the residual cells that cause relapses. A preclinical syngeneic mouse model, consisting of IV injection of 1 million of 5T33 cells in a KaLwRij C57/BL6 mouse, was treated 10 days later with an anti-mCD138 antibody, called 9E7.4, radiolabeled with astatine-211. Four activities of the 211At-9E7.4 radioimmunoconjugate were tested in two independent experiments: 370 kBq (n = 16), 555 kBq (n = 10), 740 kBq (n = 17) and 1100 kBq (n = 6). An isotype control was also tested at 555 kBq (n = 10). Biodistribution, survival rate, hematological parameters, enzymatic hepatic toxicity, histological examination and organ dosimetry were considered. The survival median of untreated mice was 45 days after engraftment. While the activity of 1100 kBq was highly toxic, the activity of 740 kBq offered the best efficacy with 65% of overall survival 150 days after the treatment with no evident sign of toxicity. This work demonstrates the pertinence of treating minimal residual disease of multiple myeloma with an anti-CD138 antibody coupled to astatine-211.

Investigation on the reactivity of nucleophilic radiohalogens with arylboronic acids in water: access to an efficient single-step method for the radioiodination and astatination of antibodies.

Easy access to radioiodinated and 211At-labelled bio(macro)molecules is essential to develop new strategies in nuclear imaging and targeted radionuclide therapy of cancers. Yet, the labelling of complex molecules with heavy radiohalogens is often poorly effective due to the multiple steps and intermediate purifications needed. Herein, we investigate the potential of arylboron chemistry as an alternative approach for the late stage labelling of antibodies. The reactivity of a model precursor, 4-chlorobenzeneboronic acid (1) with nucleophilic iodine-125 and astatine-211 was at first investigated in aqueous conditions. In the presence of a copper(ii) catalyst and 1,10-phenanthroline, quantitative radiochemical yields (RCYs) were achieved within 30 minutes at room temperature. The optimum conditions were then applied to a CD138 targeting monoclonal antibody (mAb) that has previously been validated for imaging and therapy in a preclinical model of multiple myeloma. RCYs remained high (>80% for 125I-labelling and >95% for 211At-labelling), and the whole procedure led to increased specific activities within less time in comparison with previously reported methods. Biodistribution study in mice indicated that targeting properties of the radiolabelled mAb were well preserved, leading to a high tumour uptake in a CD138 expressing tumour model. The possibility of divergent synthesis from a common modified carrier protein demonstrated herein opens facilitated perspectives in radiotheranostic applications with the radioiodine/211At pairs. Overall, the possibility to develop radiolabelling kits offered by this procedure should facilitate its translation to clinical applications.

Cell Tracking in Cancer Immunotherapy.

The impressive development of cancer immunotherapy in the last few years originates from a more precise understanding of control mechanisms in the immune system leading to the discovery of new targets and new therapeutic tools. Since different stages of disease progression elicit different local and systemic inflammatory responses, the ability to longitudinally interrogate the migration and expansion of immune cells throughout the whole body will greatly facilitate disease characterization and guide selection of appropriate treatment regiments. While using radiolabeled white blood cells to detect inflammatory lesions has been a classical nuclear medicine technique for years, new non-invasive methods for monitoring the distribution and migration of biologically active cells in living organisms have emerged. They are designed to improve detection sensitivity and allow for a better preservation of cell activity and integrity. These methods include the monitoring of therapeutic cells but also of all cells related to a specific disease or therapeutic approach. Labeling of therapeutic cells for imaging may be performed in vitro, with some limitations on sensitivity and duration of observation. Alternatively, in vivo cell tracking may be performed by genetically engineering cells or mice so that may be revealed through imaging. In addition, SPECT or PET imaging based on monoclonal antibodies has been used to detect tumors in the human body for years. They may be used to detect and quantify the presence of specific cells within cancer lesions. These methods have been the object of several recent reviews that have concentrated on technical aspects, stressing the differences between direct and indirect labeling. They are briefly described here by distinguishing ex vivo (labeling cells with paramagnetic, radioactive, or fluorescent tracers) and in vivo (in vivo capture of injected radioactive, fluorescent or luminescent tracers, or by using labeled antibodies, ligands, or pre-targeted clickable substrates) imaging methods. This review focuses on cell tracking in specific therapeutic applications, namely cell therapy, and particularly CAR (Chimeric Antigen Receptor) T-cell therapy, which is a fast-growing research field with various therapeutic indications. The potential impact of imaging on the progress of these new therapeutic modalities is discussed.

Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes.

BACKGROUND: Genome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far, PDCD1 editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments. METHODS: Here we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to edit PDCD1 gene in human effector memory CD8+ T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validated PDCD1 editing through sequencing and cytometry in each T cell clone, together with T-cell receptor (TCR) chain's sequencing. We also performed whole transcriptomic analyses on wild-type (WT) and edited T cell clones. Finally, we documented in vitro and in vivo through adoptive transfer in NOD scid gamma (NSG) mice, the antitumor properties of WT and PD-1KO T cell clones, expressing the same TCR. RESULTS: Here we demonstrated the feasibility to edit PDCD1 gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent on PDCD1-edited T cell clones. Extensive characterization of a panel of T cell clones expressing the same TCR and exhibiting similar functional avidity demonstrated superior antitumor reactivity against a PD-L1 expressing melanoma cell line. Transcriptomic analysis revealed a downregulation of genes involved in proliferation and DNA replication in PD-1-deficient T cell clones, whereas genes involved in metabolism and cell signaling were upregulated. Finally, we documented the superior ability of PD-1-deficient T cells to significantly delay the growth of a PD-L1 expressing human melanoma tumor in an NSG mouse model. CONCLUSION: The use of such lymphocytes for adoptive cell transfer purposes, associated with other approaches modulating the tumor microenvironment, would be a promising alternative to improve immunotherapy efficacy in solid tumors.

Molecular screening of interleukin-6 gene promoter and influence of -174G/C polymorphism on breast cancer.

Interleukin-6 (IL-6) is a cytokine involved in different physiologic and pathophysiologic processes including carcinogenesis. In 2003, a single nucleotide polymorphism (-174G/C) of the IL-6 gene promoter has been linked to breast cancer prognosis in node-positive (N+) breast cancer patients. Since, different studies have led to conflicting conclusions about its role as a prognostic and/or diagnostic marker. The primary aim of our study was to investigate the link between -174G/C polymorphism and breast cancer risk on the one hand, and -174G/C polymorphism and prognosis in different groups of patients: sporadic N+breast cancers (n=138), sporadic N- breast cancers (n=95) and familial breast cancer (n=60) on the other hand. The variables of interest were disease-free survival and overall survival. The secondary aim of the study was to screen IL-6 gene promoter using direct sequencing to identify new polymorphisms in our French Caucasian breast cancer population. No association or trend of association between -174G/C polymorphism of IL-6 gene promoter gene and breast cancer diagnosis or prognosis was shown, even in meta-analyses. Furthermore, we have identified four novel polymorphic sites in the IL-6 gene promoter region: -764G-->A, -757C-->T, -233T-->A, 15C-->A.

[Alpha-Radioimmunotherapy: principle and relevance in anti-tumor immunity]. / Radio-immunothérapie alpha - Principes et intérêts en immunité antitumorale.

Alpha-radioimmunotherapy (α-RIT) is a targeted anti-tumor therapy using usually a monoclonal antibody specific for a tumor antigen that is coupled to an α-particle emitter. α-emitters represent an ideal tool to eradicate disseminated tumors or metastases. Recent data demonstrate that ionizing radiation in addition to its direct cytotoxic ability can also induce an efficient anti-tumor immunity. This suggests that biologic effects on irradiated tissues could be used to potentiate immunotherapy efficacy and opens the way for development of new therapies combining α-RIT and different types of immunotherapy.

Radioimmunotherapy for Treatment of Acute Leukemia.

Acute leukemias are characterized by accumulation of immature cells (blasts) and reduced production of healthy hematopoietic elements. According to the lineage origin, two major leukemias can be distinguished: acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Although the survival rate for pediatric ALL is close to 90%, half of the young adults with AML or ALL and approximately 90% of older patients with AML or ALL still die of their disease, raising the need for innovative therapeutic approaches. As almost all leukemic blasts express specific surface antigens, targeted immunotherapy appears to be particularly promising. However, published results of immunotherapy alone are generally modest. Radioimmunotherapy (RIT) brings additional therapeutic mechanisms using radiolabeled monoclonal antibodies (mAbs) directed to tumor antigens, thus adding radiobiological cytotoxicity to immunologic cytotoxicity. Because of the high radiosensitivity of tumor cells and the diffuse widespread nature of the disease, making it rapidly accessible to circulating radiolabeled mAbs, acute leukemias represent relevant indications for RIT. With the development of recombinant and humanized mAbs, innovative radionuclides, and more efficient radiolabeling and pretargeting techniques, RIT has significantly improved over the last 10 years. Different approaches of α and ß RIT targeting CD22, CD33, CD45, or CD66 antigens have already been evaluated or are currently being developed in the treatment of acute leukemia. This review summarizes the preclinical and clinical studies demonstrating the potential of RIT in treatment of AML and ALL.

Single-Dose Anti-CD138 Radioimmunotherapy: Bismuth-213 is More Efficient than Lutetium-177 for Treatment of Multiple Myeloma in a Preclinical Model.

OBJECTIVES: Radioimmunotherapy (RIT) has emerged as a potential treatment option for multiple myeloma (MM). In humans, a dosimetry study recently showed the relevance of RIT using an antibody targeting the CD138 antigen. The therapeutic efficacy of RIT using an anti-CD138 antibody coupled to (213)Bi, an α-emitter, was also demonstrated in a preclinical MM model. Since then, RIT with ß-emitters has shown efficacy in treating hematologic cancer. In this paper, we investigate the therapeutic efficacy of RIT in the 5T33 murine MM model using a new anti-CD138 monoclonal antibody labeled either with (213)Bi for α-RIT or (177)Lu for ß-RIT. METHODS: A new monoclonal anti-CD138 antibody, 9E7.4, was generated by immunizing a rat with a murine CD138-derived peptide. Antibody specificity was validated by flow cytometry, biodistribution, and α-RIT studies. Then, a ß-RIT dose-escalation assay with the (177)Lu-radiolabeled 9E7.4 mAb was performed in KalwRij C57/BL6 mice 10 days after i.v. engraftment with 5T33 MM cells. Animal survival and toxicological parameters were assessed to define the optimal activity. RESULTS: α-RIT performed with 3.7 MBq of (213)Bi-labeled 9E7.4 anti-CD138 mAb increased median survival to 80 days compared to 37 days for the untreated control and effected cure in 45% of animals. ß-RIT performed with 18.5 MBq of (177)Lu-labeled 9E7.4 mAb was well tolerated and significantly increased mouse survival (54 vs. 37 days in the control group); however, no mice were cured with this treatment. CONCLUSION: This study revealed the advantages of α-RIT in the treatment of MM in a preclinical model where ß-RIT shows almost no efficacy.

Alpha Particles Induce Autophagy in Multiple Myeloma Cells.

OBJECTIVES: Radiation emitted by the radionuclides in radioimmunotherapy (RIT) approaches induce direct killing of the targeted cells as well as indirect killing through the bystander effect. Our research group is dedicated to the development of α-RIT, i.e., RIT using α-particles especially for the treatment of multiple myeloma (MM). γ-irradiation and ß-irradiation have been shown to trigger apoptosis in tumor cells. Cell death mode induced by (213)Bi α-irradiation appears more controversial. We therefore decided to investigate the effects of (213)Bi on MM cell radiobiology, notably cell death mechanisms as well as tumor cell immunogenicity after irradiation. METHODS: Murine 5T33 and human LP-1 MM cell lines were used to study the effects of such α-particles. We first examined the effects of (213)Bi on proliferation rate, double-strand DNA breaks, cell cycle, and cell death. Then, we investigated autophagy after (213)Bi irradiation. Finally, a coculture of dendritic cells (DCs) with irradiated tumor cells or their culture media was performed to test whether it would induce DC activation. RESULTS: We showed that (213)Bi induces DNA double-strand breaks, cell cycle arrest, and autophagy in both cell lines, but we detected only slight levels of early apoptosis within the 120 h following irradiation in 5T33 and LP-1. Inhibition of autophagy prevented (213)Bi-induced inhibition of proliferation in LP-1 suggesting that this mechanism is involved in cell death after irradiation. We then assessed the immunogenicity of irradiated cells and found that irradiated LP-1 can activate DC through the secretion of soluble factor(s); however, no increase in membrane or extracellular expression of danger-associated molecular patterns was observed after irradiation. CONCLUSION: This study demonstrates that (213)Bi induces mainly necrosis in MM cells, low levels of apoptosis, and autophagy that might be involved in tumor cell death.

Combining α-Radioimmunotherapy and Adoptive T Cell Therapy to Potentiate Tumor Destruction.

Ionizing radiation induces direct and indirect killing of cancer cells and for long has been considered as immunosuppressive. However, this concept has evolved over the past few years with the demonstration that irradiation can increase tumor immunogenicity and can actually favor the implementation of an immune response against tumor cells. Adoptive T-cell transfer (ACT) is also used to treat cancer and several studies have shown that the efficacy of this immunotherapy was enhanced when combined with radiation therapy. α-Radioimmunotherapy (α-RIT) is a type of internal radiotherapy which is currently under development to treat disseminated tumors. α-particles are indeed highly efficient to destroy small cluster of cancer cells with minimal impact on surrounding healthy tissues. We thus hypothesized that, in the setting of α-RIT, an immunotherapy like ACT, could benefit from the immune context induced by irradiation. Hence, we decided to further investigate the possibilities to promote an efficient and long-lasting anti-tumor response by combining α-RIT and ACT. To perform such study we set up a multiple myeloma murine model which express the tumor antigen CD138 and ovalbumine (OVA). Then we evaluated the therapeutic efficacy in the mice treated with α-RIT, using an anti-CD138 antibody coupled to bismuth-213, followed by an adoptive transfer of OVA-specific CD8+ T cells (OT-I CD8+ T cells). We observed a significant tumor growth control and an improved survival in the animals treated with the combined treatment. These results demonstrate the efficacy of combining α-RIT and ACT in the MM model we established.