Development and preclinical characterization of anti-CD38 Affitins for positron emission tomography imaging of multiple myeloma

Le myélome multiple représente 10% des hémopathies malignes et demeure encore aujourd'hui incurable malgré les progrès réalisés au cours des deux dernières décennies. Dans ce contexte, le développement de nouvelles stratégies de vectorisation constitue un axe de recherche majeur. Les anticorps sont les molécules les plus utilisées pour le ciblage moléculaire mais leurs caractéristiques pharmacocinétiques et structurelles rendent leur usage non optimal pour des applications d’imagerie. Les Affitines sont des protéines d’affinité artificielle de faible poids moléculaire montrant une affinité et une spécificité comparables à celles des anticorps tout en présentant une stabilité accrue. Ces différentes caractéristiques font de ces protéines d’excellentes candidates à une utilisation en imagerie en alternative aux anticorps. Le but de ce travail de thèse était donc d’investiguer l’intérêt de cette nouvelle classe de vecteur pour l’imagerie phénotypique du myélome multiple. La première partie de cette étude a été consacrée au développement et à la caractérisation d’Affitines spécifiques de CD38, une glycoprotéine surexprimée par les plasmocytes malins. Ce travail est toujours en cours et sera poursuivi par l’évaluation préclinique des Affitines anti-CD38 identifiées. La seconde partie du doctorat a été consacrée à une étude d’imagerie basée sur l’utilisation d’un anticorps anti-CD38 radiomarqué au cuivre 64. L’objectif principal de cette étude était d’apporter un premier éclairage sur le ciblage du CD38 pour l’imagerie et de servir de point de comparaison avec les études similaires qui seront menées avec les Affitines anti-CD38 générées.Multiple myeloma accounts for 10% of hematological malignancies and still remains incurable despite medical progress. In this context, new targeting strategies represent a major step forward which can improve patient’s diagnosis and therapeutic monitoring. Antibodies are the most widely used targeting molecules but their pharmacokinetic and structural characteristics make their use suboptimal for imaging applications. Affitins are artificial affinity proteins with low molecular weight that exhibit comparable affinity and specificity to those of antibodies while being highly stable. These different characteristics make Affitins excellent candidates for a use in imaging as an alternative to antibodies. The aim of this thesis was therefore to investigate the interest of this new class of vector for phenotypic imaging of multiple myeloma. The first part of this work was devoted to the development and characterization of Affitins specific for CD38, a glycoprotein overexpressed by multiple myeloma cells. This study is still ongoing and will be followed by the preclinical evaluation of anti-CD38 Affitins. The second part of the thesis work was devoted to an imaging study based on the use of an anti- CD38 antibody radiolabelled with copper 64. The main objective of this study was to shed a first light on CD38 targeting for imaging and to serve as a point of comparison with similar studies that will be conducted with the anti- CD38 Affitins previously generated

A lineage-specific methylation pattern controls the transcription of the polycistronic mRNA coding MELOE melanoma antigens

International audienceWe recently characterized two melanoma antigens MELOE-1 and MELOE-2 derived from a polycistronic RNA overexpressed in the melanocytic lineage. This transcription profile was because of hypomethylation of the meloe proximal promoter in melanomas and melanocytes. Here, we investigated whether this demethylation was restricted to the meloe promoter or was linked to a general lack of methylation at the meloe locus in the melanocytic lineage. We established the methylation pattern of the locus spanning more than 40 kbp, focusing on CpG islands, using DNA bisulfite conversion and pyrosequencing. The study was carried out on cultured cell lines (melanoma, melanocyte, colon cancer, and mesothelioma cell lines), healthy tissues (skin and colon), and melanoma tumors. Demethylation, specifically observed in the melanocytic lineage, involves a large promoter area and not the entire meloe locus. This enables updating a tight regulation of meloe transcription in this lineage, suggesting tissue-specific epigenetic mechanisms. Associated with the previously described translational mechanisms, leading to the specific expression of MELOE-1 and MELOE-2 in melanomas, this makes MELOE-derived antigens a relevant candidate for immunotherapy of melanoma. Melanoma Res 00:000–00

How Sublaminar Bands Affect Postoperative Sagittal Alignment in AIS Patients with Preoperative Hypokyphosis? Results of a Series of 34 Patients with 2-Year Follow-Up

Hypokyphosis is currently observed in thoracic idiopathic scoliosis. The use of sublaminar bands allows a good restoration of sagittal balance of the spine. The aim of the study was to provide a middle-term radiographic analysis of patients with adolescent idiopathic scoliosis with preoperative hypokyphosis treated by posterior arthrodesis with sublaminar bands. This retrospective study included 34 patients with Lenke 1 scoliosis associated with hypokyphosis (TK < 20°). A radiographic evaluation was performed with a 2-year follow-up. Cobb angle, cervical lordosis, thoracic kyphosis, lumbar lordosis, and pelvic parameters were measured preoperatively, postoperatively, and at 6-month and 2-year follow-up. The mean preoperative thoracic kyphosis was 10.5° versus 24.1° postoperatively (p<0.001), representing a mean gain of 13°. Cobb angle ranged from 59.3° to 17.9° postoperatively (mean correction 69%, p<0.001). Cobb angle increased between the immediate postoperative measurement and the 6-month follow-up (17.9 versus 19.9, p=0.03). Cervical curvature changed from a 5.6° kyphosis to a 3.5° lordosis (p=0.001). Concerning lumbar lordosis, preoperative measurement was 39.7° versus 41.3° postoperatively (p=0.27). At 6-month follow-up, lumbar lordosis significantly increased to 43.6° (p=0.03). All parameters were stable at final follow-up. Correction performed by sublaminar bands is efficient for both fontal and sagittal planes. Moreover, the restoration of normal thoracic kyphosis is followed by an adaptation of the adjacent curvatures with improved cervical lordosis and lumbar lordosis

Deep mutational engineering of broadly-neutralizing nanobodies accommodating SARS-CoV-1 and 2 antigenic drift

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ImmunoPET in Multiple Myeloma—What? So What? Now What?

International audienceDespite constant progress over the past three decades, multiple myeloma (MM) is still an incurable disease, and the identification of new biomarkers to better select patients and adapt therapy is more relevant than ever. Recently, the introduction of therapeutic monoclonal antibodies (mAbs) (including direct-targeting mAbs and immune checkpoint inhibitors) appears to have changed the paradigm of MM management, emphasizing the opportunity to cure MM patients through an immunotherapeutic approach. In this context, immuno-positron emission tomography (immunoPET), combining the high sensitivity and resolution of a PET camera with the specificity of a radiolabelled mAb, holds the capability to cement this new treatment paradigm for MM patients. It has the potential to non-invasively monitor the distribution of therapeutic antibodies or directly monitor biomarkers on MM cells, and to allow direct observation of potential changes over time and in response to various therapeutic interventions. Tumor response could, in the future, be anticipated more effectively to provide individualized treatment plans tailored to patients according to their unique imaging signatures. This work explores the important role played by immunotherapeutics in the management of MM, and focuses on some of the challenges for this drug class and the significant interest of companion imaging agents such as immunoPET

Efficacy of Astatine-211 radioimmunotherapy of Multiple Myeloma using an anti-mCD138 monoclonal antibody in a syngeneic murine model

Abstracts of Annual Congress of the European Association of Nuclear Medicine October 13 – 17, 2018 Düsseldorf, GermanyInternational audienceAim: Multiple myeloma is a B-cell malignancy of terminally differentiated plasma cells within the bone marrow. In spite of a very active search for new treatments, cure is almost never achieved. Alpha-radioimmunotherapy (RIT) is a new cancer treatment modality with tumour specific antibodies coupled to alpha particle-emitting radionuclides. CD138 (Syndecan-1) is found mainly in epithelial cells, but was shown to be expressed by most myeloma cells, both in human and in the mouse. The aim of the study was to evaluate the biodistribution, toxicity and efficacy of a rat Astatin-211-labelled anti-mouse CD138 antibody (211At-9E7.4) in a syngeneic mouse myeloma model. Materials and methods: C57BL/KaLwRij mice were grafted with 106 5T33 cells (murine myeloma cell line). Biodistribution was studied 15 min, 1h, 4h, 7h, 14h and 21h post-administration of 211At- 9E7.4 mAb. Toxicity (animal weight, blood cell counts and transaminase) and RIT efficacy were studied after a dose escalation using 370, 555, 740 and 1110 kBq of 211At-9E7.4 and two control groups 211At-IgG2a isotype control at 555 kBq or no treatment, 10 days after tumour engraftment. Results: Studies demonstrated a highly statistical survival benefit for the mice treated with 211At-9E7.4 at 555 kBq (p=0,0006) and 740 kBq (p<0,0001). At 555 kBq, the survival median was increase by 34 days and at 740 kBq 65% of the mice survived 160 days after engraftment. For treatments with 370 kBq with 211At-9E7.4 or 211At-isotype control at 555 kBq no significant benefit was observed. The higher activity with 1110 kBq of 211At-9E7.4 was clearly radiotoxic since mice were euthanized after a lost more than 30% of baseline weight 14 days after radiopharmaceutical injection. For the other groups, except transient decreases of leukocytes and red cells, no other toxicity could be demonstrated, especially on liver function, which does not seem to be affected. Concerning red blood cells, the effect is much weaker than that observed with the use of Bismuth-213 at an injected activity of 3.7 MBq. Conclusions: RIT of MM using Astatine-211 coupled to monoclonal antibody directed against murine CD138 is effective. The activity in astate-211 which allows 60% survival corresponds to an activity injected in Bismuth-213 located between 3.7 and 7.4 MBq, which seems to reflect the influence of the half life of the two radionuclides. In addition, the upper half-life of astatine appears to be a benefit, particularly because of the lower toxicity observed in this syngeneic model of MM

Biodistribution, Imaging and Metabolism Studies of 64-Copper Radiolabelled Monoclonal Antibody : Comparison of DOTA and TE1PA Chelating Agents in a Murine Model of Multiple Myeloma

International audiencePurpose: TE1PA is a C-functionalized monopicolinate cyclam designed for 64-copper chelation. It was proven to have excellent Cu(II) and Cu(I) chelation properties, with fast kinetics, high thermodynamic stability and resistance to transchelation in vitro, and a usefulness for phenotypic imagery when coupled to antibodies. This work presents in vivo studies (biodistribution, metabolism and imaging) of TE1PA in a syngenic multiple myeloma model on mice, compared to DOTA, its main competitor. Materials and methods: p-SCN-Bn-TE1PA and DOTA-NHS ester were grafted on 9E7.4 rat IgG2a antibody, targeting murine CD-138. Immunoconjugates were then radiolabelled with 64Cu. Both 64Cu-9E7.4-p-SCN-Bn-TE1PA and 64Cu-9E7.4-NHS-DOTA were injected in 12 mice previously grafted with 5T33 cells, allowing the development of subcutaneous tumors expressing CD-138. Each mouse was injected with 100 μg of radioimmunoconjugates corresponding to 10 MBq of 64Cu. For each time studied (2h, 24h and 48h post-injection), a biodistribution and a liver metabolism studies were conducted on mice for both radiolabelled antibodies. Additionally, a micro-PET scan was performed on 4 mice injected with 64Cu-9E7.4-p-SCN-Bn-TE1PA at those 3 times. Results: Biodistribution study shows an excellent hepatic clearance of the 64Cu-9E7.4-p-SCN-Bn-TE1PA over time. Significantly higher radioactivity was found in blood, lungs and heart at 48h PI for 64Cu-9E7.4-NHS-DOTA, which suggests a release of 64Cu from antibody, in parallel with a higher intestinal elimination. This was correlated by the liver metabolism study, which shows a better in vivo resistance to transchelation for 64Cu-9E7.4-p-SCN-Bn-TE1PA. The imaging study of 64Cu-9E7.4-p-SCN-Bn-TE1PA shows an increasing targeting of the tumors over time and confirms the hepatic clearance at 24h and 48h PI. Conclusion: 64Cu-9E7.4-p-NCS-Bn-TE1PA has shown a very high tumor targeting, associated to an increasing of tumor-to-tissues ratio over time that suggests an overall clearance from healthy tissues. Compared to DOTA, TE1PA displays an in vivo stability and resistance to transchelation significantly superior. This confirms the previous results obtained in vitro and 64Cu-labelled TE-1PA proved once again its usefulness for immuno-PET imaging in a preclinical model

Comparison of Immuno-PET of CD138 and PET imaging with 64CuCl2 and 18F-FDG in a preclinical syngeneic model of multiple myeloma

International audiencePurpose:Although recent data from the literature suggest that PET imaging with [18]-Fluorodeoxyglucose (18F-FDG) is a promising technique in multiple myeloma (MM), the development of other radiopharmaceuticals seems relevant. CD138 is currently used as a standard marker in many laboratories for the identification and purification of myeloma cells, and could be used in phenotype tumor imaging. In this study, we evaluated a 64Cu-labeled anti-CD138 murine antibody (64Cu-TE2A-9E7.4) and a metabolic tracer (64CuCl2) for PET imaging in a MM syngeneic mouse model.Experimental Design and Results:64Cu-TE2A-9E7.4 antibody and 64CuCl2 were evaluated via PET imaging and biodistribution studies in C57BL / KaLwRij mice bearing either 5T33-MM subcutaneous tumors or bone lesions. These results were compared to 18F-FDG-PET imaging. Autoradiography and histology of representative tumors were secondly conducted. In biodistribution and PET studies, 64Cu-TE2A-9E7.4 displayed good tumor uptake of subcutaneous and intra-medullary lesions, greater than that demonstrated with 18F-FDG-PET. In control experiments, only low-level, non-specific uptake of 64Cu-labeled isotype IgG was observed in tumors. Similarly, low activity concentrations of 64CuCl2 were accumulated in MM lesions. Histopathologic analysis of the immuno-PET-positive lesions revealed the presence of plasma cell infiltrates within the bone marrow.Conclusions:64Cu-labeled anti-CD138 antibody can detect subcutaneous MM tumors and bone marrow lesions with high sensitivity, outperforming 18F-FDG-PET and 64CuCl2 in this preclinical model. These data support 64Cu-anti-CD138 antibody as a specific and promising new imaging radiopharmaceutical agent in MM

PET Imaging of Multiple Myeloma : Comparison of 89Zr- and 64Cu-labeled anti-CD138 Conjugates to 64CuCl2 and 18F-FDG in a Preclinical Syngeneic Model

Abstracts of Annual Congress of the European Association of Nuclear Medicine October 13 – 17, 2018 Düsseldorf, GermanyInternational audiencePurpose: Although recent data from the literature suggest that PET imaging with [18]-Fluorodeoxyglucose (18F-FDG) is a promising technique in multiple myeloma (MM), the development of other radiopharmaceuticals seems relevant. CD138 is currently used as a standard marker in many laboratories for the identification and purification of myeloma cells, and could be used in phenotype tumor imaging. In this study, we evaluated 2 conjugates of an anti-CD138 murine antibody (9E7.4) and compared them to metabolic tracers (64CuCl2and 18F-FDG) for PET imaging in a MM syngeneic mouse model. Subjects and Methods: 9E7.4 was conjugated to TE2A-benzyl isothiocyanate (TE2A) and desferrioxamine (DFO) chelators for copper-64 (64Cu) and zirconium- 89 (89Zr) labeling. 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 antibodies and 64CuCl2 were evaluated via PET imaging and biodistribution studies in C57BL / KaLwRij mice bearing either 5T33-MM subcutaneous tumors or bone lesions. These results were compared to 18F-FDG-PET imaging. Autoradiography and histology of representative tumors were secondly conducted. Results: In biodistribution and PET studies, 64Cu-TE2A-9E7.4 and89Zr-DFO-9E7.4displayed comparable good tumor uptake of subcutaneous tumors. On the opposite, only low-level concentrations of 64CuCl2 were accumulated in MM lesions. PET/CT imaging of the disseminated model with 64Cu TE2A-9E7.4 and 89Zr-DFO-9E7.4 showed high uptake of the probes at the site of intra-medullary lesions, greater than that demonstrated with 18F-FDG-PET and correlating with the bioluminescence imaging of the tumor. Histopathologic analysis of the immuno-PET-positive lesions also confirmed the presence of plasma cell infiltrates within the bone marrow. Comparison of both 9E7.4 conjugates revealed higher non specific bone uptakes of 89Zr-DFO-9E7.4 than 64Cu-TE2A-9E7.4 (3.1±1.15 vs 1.48±0.29 respectively at 24h PI; p=0.0061; non-parametric test) while the opposite was observed for tumor-to-blood ratio (1.42±0.24 vs 4.08±1.09 respectively at 24h PI; p=0.0391; non-parametric test). Such observations were consistent with the known in vivo gradual transchelation of 89Zr over time which could reduce the efficacy of a 89Zr-labeled immuno-PET probe as an effective tool for bone lesions imaging. Conclusion: 64Cu- and 89Zr-labeled anti-CD138 antibody can detect subcutaneous MM tumors and bone marrow lesions with high sensitivity, outperforming 18F-FDG-PET and 64CuCl2 in this preclinical model. 64Cu- anti-CD138 antibody had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a specific and promising new imaging radiopharmaceutical agent in MM

What is the Best Radionuclide for Immuno-PET of Multiple Myeloma? A Comparison Study Between ⁸⁹Zr- and ⁶⁴Cu-Labeled Anti-CD138 in a Preclinical Syngeneic Model

Although positron emission tomography (PET) imaging with 18-Fluorodeoxyglucose (18F-FDG) is a promising technique in multiple myeloma (MM), the development of other radiopharmaceuticals seems relevant. CD138 is currently used as a standard marker for the identification of myeloma cells and could be used in phenotype tumor imaging. In this study, we used an anti-CD138 murine antibody (9E7.4) radiolabeled with copper-64 (64Cu) or zirconium-89 (89Zr) and compared them in a syngeneic mouse model to select the optimal tracers for MM PET imaging. Then, 9E7.4 was conjugated to TE2A-benzyl isothiocyanate (TE2A) and desferrioxamine (DFO) chelators for 64Cu and 89Zr labeling, respectively. 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 antibodies were evaluated by PET imaging and biodistribution studies in C57BL/KaLwRij mice bearing either 5T33-MM subcutaneous tumors or bone lesions and were compared to 18F-FDG-PET imaging. In biodistribution and PET studies, 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 displayed comparable good tumor uptake of subcutaneous tumors. On the bone lesions, PET imaging with 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 showed higher uptake than with 18F-FDG-PET. Comparison of both 9E7.4 conjugates revealed higher nonspecific bone uptakes of 89Zr-DFO-9E7.4 than 64Cu-TE2A-9E7.4. Because of free 89Zr&#8217;s tropism for bone when using 89Zr-anti-CD138, 64Cu-anti-CD138 antibody had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a specific new imaging radiopharmaceutical agent in MM