Syndecan-1 antigen, a promising new target for triple-negative breast cancer immuno-PET and radioimmunotherapy. A preclinical study on MDA-MB-468 xenograft tumors

International audienceBackgroundOverexpression of syndecan-1 (CD138) in breast carcinoma correlates with a poor prognosis and an aggressive phenotype. The objective of this study was to evaluate the potential of targeting CD138 by immuno-PET imaging and radioimmunotherapy (RIT) using the antihuman syndecan-1 B-B4 mAb radiolabeled with either 124I or 131I in nude mice engrafted with the triple-negative MDA-MB-468 breast cancer cell line.MethodThe immunoreactivity of 125I-B-B4 (80%) was determined, and the affinity of 125I-B-B4 and the expression of CD138 on MDA-MB-468 was measured in vitro by Scatchard analysis. CD138 expression on established tumors was confirmed by immunohistochemistry. A biodistribution study was performed in mice with subcutaneous MDA-MB-468 and 125I-B-B4 at 4, 24, 48, 72, and 96 h after injection and compared with an isotype-matched control. Tumor uptake of B-B4 was evaluated in vivo by immuno-PET imaging using the 124I-B-B4 mAb. The maximum tolerated dose (MTD) was determined from mice treated with 131I-B-B4 and the RIT efficacy evaluated.Results 125I-B-B4 affinity was in the nanomolar range (Kd = 4.39 ± 1.10 nM). CD138 expression on MDA-MB-468 cells was quite low (Bmax = 1.19 × 104 ± 9.27 × 102 epitopes/cell) but all expressed CD138 in vivo as determined by immunohistochemistry. The tumor uptake of 125I-B-B4 peaked at 14% injected dose (ID) per gram at 24 h and was higher than that of the isotype-matched control mAb (5% ID per gram at 24 h). Immuno-PET performed with 124I-B-B4 on tumor-bearing mice confirmed the specificity of B-B4 uptake and its retention within the tumor. The MTD was reached at 22.2 MBq. All mice treated with RIT (n = 8) as a single treatment at the MTD experienced a partial (n = 3) or complete (n = 5) response, with three of them remaining tumor-free 95 days after treatment.ConclusionThese results demonstrate that RIT with 131I-B-B4 could be considered for the treatment of metastatic triple-negative breast cancer that cannot benefit from hormone therapy or anti-Her2/neu immunotherapy. Immuno-PET for visualizing CD138-expressing tumors with 124I-B-B4 reinforces the interest of this mAb for diagnosis and quantitative imaging

Caractérisation de la réponse T cytotoxique à l'aide d'anticorps spécifiques de complexes HLA-A2/Mage3 de tumeur et ciblage des cellules cancéreuses en radioimmunothérapie alpha in vitro

Les lymphocytes T de patients atteints de cancers sont capables de reconnaître les cellules tumorales et de les détruire lorsqu'ils sont stimulés de façon appropriée. Une activité importante de recherche vise à établir les conditions optimales de cette activation et à caractériser les complexes CMH/peptides reconnus. Parmi ces complexes, ceux issus de la famille des " cancer testis antigenes " ont une expression strictement restreinte aux cellules tumorales. Nous avons isolé des anticorps monoclonaux de souris dirigés contre le complexe HLA-A2/Mage3. Ces anticorps nous ont permis de mesurer les densités de complexes exprimées à la surface des cellules, permettant de définir le nombre d'antigènes nécessaires à la réponse cytotoxique de lymphocytes T CD8 spécifiques. D'autre part nous avons évalué la possibilité de cibler ces complexes en radioimmunothérapie à l'aide de radio émetteur alpha dont le fort potentiel radiotoxique permet de compenser la faible expression de surface des complexes CMH/peptide.When stimulated appropriately, the T lymphocytes of cancer patients are able to recognise and destroy tumor cells. A major goal of research in this field is to establish optimal conditions for this T cell response and to characterise the MHC/peptide complexes recognised. Among these complexes, those derived from the family of cancer testis antigens have an expression that is restricted to tumoral cells. We have identified murine monoclonal antibodies directed against the HLA-A2/Mage3 complex. Using these antibodies, we have been able to measure the density of complexes expressed at the cell surface, thus enabling definition of the number of antigens necessary to initiate a cytotoxic response by specific CD8 T lymphocytes. We have additionally evaluated the possibility of targeting these complexes by radioimmunotherapy using an alpha radio emitter whose powerful radiotoxic potential compensates the weak surface expression of MHC/peptide complexes.NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

A fast and efficient HLA multimer-based sorting procedure that induces little apoptosis to isolate clinical grade human tumor specific T lymphocytes

International audienceHLA multimers are now widely used to stain and sort CD8 T lymphocytes speciWc for epitopes from viral or tumoral antigens presented in an HLA class I context. However, the transfer of this technology to a clinical setting to obtain clinical grade CD8 T lymphocytes that may be used in adoptive cell transfer (ACT) is hindered by two main obstacles: the Wrst obstacle is the use of streptavi-din or derived products that are not available in clinical grade to multimerize HLA/peptide monomers and the second is the reported high degree of apoptosis that eventually occurs when T cell receptors are crosslinked by HLA multi-mers. In the present report, we describe new HLA multi-mers composed of immunomagnetic beads covalently coupled to a mAb speciWc for the AviTag peptide and coated with HLA/peptide monomers bearing the non bio-tinylated AviTag at the COOH terminus of the HLA heavy chain. Thus, all the components of this new reagent can be obtained in clinical grade. We compared these new multi-mers with the previously described multimers made with streptavidin beads coated with biotinylated HLA/peptide monomers, in terms of sorting eYciency, recovery of functional T cells, apoptosis and activation. We provide evidence that the new multimers could very eYciently sort pure populations of T lymphocytes speciWc for three diVer-ent melanoma antigens (Melan-A, gp100 and NA17-A) after a single peptide stimulation of melanoma patients' PBMC. The recovered speciWc T cells were cytotoxic against the relevant melanoma cell-lines and, in most cases, produced cytokines. In addition, in marked contrast with streptavidin-based multimers, our new multimers induced very little apoptosis or activation after binding speciWc T lymphocytes. Altogether, these new multimers fulWll all the necessary requirements to select clinical grade T lympho-cytes and should facilitate the development of ACT protocols in cancer patients

Soluble HLA-I/Peptide Monomers Mediate Antigen-Specific CD8 T Cell Activation through Passive Peptide Exchange with Cell-Bound HLA-I Molecules

International audienceAccumulating evidence that serum levels of soluble class I HLA molecules (sHLA-I) can, under various pathological conditions, correlate with disease stage and/or patient survival, has stimulated interest in defining whether sHLA-I can exert immunological functions. However, despite a mounting number of publications suggesting the ability of sHLA-I to affect immune effectors in vitro, the precise underlying mechanism still remains controversial. In this article, we address potential functions of both classical and nonclassical sHLA-I, using soluble recombinant HLA-I/peptide monomers, and clearly demonstrate their ability to trigger Ag-specific activation of CD8 T cells in vitro. Furthermore, we provide strong evidence that this behavior results from the passive transfer of peptides from monomers to T cell-bound HLA-I molecules, allowing for fratricide representation and activation. Hence, we proposed a unifying model of T cell activation by HLA-I/peptide monomers, reappraising the potential involvement of sHLA-I molecules in the immune response

A simple competitive assay to determine peptide affinity for HLA class II molecules: A useful tool for epitope prediction

International audienceWe have designed a cytometry-based competition assay to evaluate peptide binding to empty recombinant HLA class II molecules. The efficiency of this assay was evaluated using recombinant HLA-DP0401 molecules (HLA-DP) produced in insect cells and 13 peptides from human telomerase reverse transcriptase (hTERT). We demonstrate that our method allowed accurate measurements of peptide Ki values and can thus discriminate strong, moderate and poor HLA-DP binders. In parallel, we showed that among hTERT peptides, the most immunodominant in healthy individuals were those with moderate affinity for HLA-DP while no T cell response could be evidenced against peptides with very strong or very low affinities for HLA-DP. This strongly suggests that the precise determination of peptide affinity with our method can improve HLA class II epitope prediction

MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection

International audienceIn a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival , which is mediated by the induction of CD8+CD45RC[lo] regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.A[a]/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.A[a]/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function

A novel and efficient approach to high-throughput production of HLA-E/peptide monomer for T-cell epitope screening

International audienceOver the past two decades, there has been a great interest in the study of HLA-E-restricted αβ T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells

New synthesis of phenyl-isothiocyanate C-functionalised cyclams. Bioconjugation and 64 Cu phenotypic PET imaging studies of multiple myeloma with the te2a derivative †

International audienceAzamacrocyclic bifunctional chelating agents (BCAs) are essential for the development of radiopharma-ceuticals in nuclear medicine and we wish to prove that their bioconjugation by a function present on a carbon atom of the macrocyclic skeleton is a solution of choice to maintain their in vivo inertness. Based on our very recent methodology using a bisaminal template and selective N-alkylation approach, a new synthesis of conjugable C-functionalised teta, te2a and cb-te2a has been developed. These chelators have indeed a growing interest in nuclear medicine for positron emission tomography (PET) and radio-immunotherapy (RIT) where they show in several cases better complexation properties than dota or dota-like macrocycles, especially with 64 Cu or 67 Cu radioisotopes. Chelators are bearing an isothiocyanate grafting function introduced by C-alkylation to avoid as much as possible a critical decrease of their che-lating properties. The synthesis is very efficient and yields the targeted ligands, teta-Ph-NCS, te2a-Ph-NCS and cb-te2a-Ph-NCS without fastidious work-up and could be easily extended to other cyclam based-BCAs. The newly synthetised te2a-Ph-NCS has been conjugated to an anti mCD138 monoclonal antibody (mAb) to evaluate its in vivo behavior and potentiality as BCA and to explore a first attempt of PET-phenotypic imaging in multiple myeloma (MM). Mass spectrometry analysis of the immunoconjugate showed that up to 4 chelates were conjugated per 9E7.4 mAb. The radiolabeling yield and specific activity post-purification of the bioconjugate 9E7.4-CSN-Ph-te2a were 95 ± 2.8% and 188 ± 27 MBq mg −1 respectively and the immunoreactivity of 64 Cu-9E7.4-CSN-Ph-te2a was 81 ± 7%. Animal experiments were carried out on 5T33-Luc(+) tumor bearing mice, either in subcutaneous or orthotopic. To achieve PET imaging, mice were injected with 64 Cu-9E7.4-CNS-Ph-te2a and acquisitions were conducted 2 and 20 h post-injection (PI). A millimetric bone uptake was localised in a sacroiliac of a MM orthotopic tumor. Nonspecific uptakes were observed at 2 h PI but, unlike for the tumor, a significant decrease was observed at 20 h PI which improves the contrast of the images