François Davodeau

Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding.

Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding

Cancer radioimmunotherapy with alpha-emitting nuclides

In lymphoid malignancies and in certain solid cancers such as medullary thyroid carcinoma, somewhat mixed success has been achieved when applying radioimmunotherapy (RIT) with β-emitters for the treatment of refractory cases. The development of novel RIT with α-emitters has created new opportunities and theoretical advantages due to the high linear energy transfer (LET) and the short path length in biological tissue of α-particles. These physical properties offer the prospect of achieving selective tumoural cell killing. Thus, RIT with α-emitters appears particularly suited for the elimination of circulating single cells or cell clusters or for the treatment of micrometastases at an early stage. However, to avoid non-specific irradiation of healthy tissues, it is necessary to identify accessible tumoural targets easily and rapidly. For this purpose, a small number of α-emitters have been investigated, among which only a few have been used for in vivo preclinical studies. Another problem is the availability and cost of these radionuclides; for instance, the low cost and the development of a reliable actinium-225/bismuth-213 generator were probably determining elements in the choice of bismuth-213 in the only human trial of RIT with an α-emitter. This article reviews the literature concerning monoclonal antibodies radiolabelled with α-emitters that have been developed for possible RIT in cancer patients. The principal radio-immunoconjugates are considered, starting with physical and chemical properties of α-emitters, their mode of production, the possibilities and difficulties of labelling, in vitro studies and finally, when available, in vivo preclinical and clinical studies.

Regulation of Inhibitory and Activating Killer-Cell Ig-Like Receptor Expression Occurs in T Cells After Termination of TCR Rearrangements

A small fraction of T cells expresses killer-cell Ig-like receptors (KIR), a family of MHC class I-specific receptors that can modulate TCR-dependent activation of effector functions. Although KIR+ cells are enriched within Ag-experienced T cell subsets, the precise relationships between KIR+ and KIR− T cells and the stage of KIR induction on these lymphocytes remain unclear. In this study, we compared KIR− and KIR+ αβ T cell clones, sorted by means of the CD158b (KIR2DL2/KIR2DL3/KIR2DS2) specific mAb GL183. We isolated several pairs of CD158b+ and CD158b− αβ T cell clones sharing identical productive and nonproductive TCR transcripts. We showed that expression of functional KIR on T cells is regulated after termination of TCR rearrangements. Transcriptional regulation of KIR genes was documented in multiple T cell clones generated from the same donor, and the presence of KIR transcripts was also detected in KIR− T cells. These results document a complex regulation of KIR expression in T cells at both pre and posttranscriptional levels, under the control of yet undefined signals provided in vivo.

Frequent Contribution of T Cell Clonotypes with Public TCR Features to the Chronic Response Against a Dominant EBV-Derived Epitope: Application to Direct Detection of Their Molecular Imprint on the Human Peripheral T Cell Repertoire

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR β-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vβ subsets (Vβ2, Vβ4, and Vβ16 positive) with highly conserved TCRβ complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR β-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR α-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR α-chain vs TCR β-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.

Diverse CD1d-restricted reactivity patterns of human T cells bearing "invariant" AV24BV11 TCR

Human and murine natural T (NT) cells, also referred to as NK1.1+ or NK T cells, express TCR with homologous V regions (hAV24/BV11 and mAV14/BV8, respectively) and conserved “invariant” TCR AVAJ junctional sequences, suggesting recognition of closely related antigens. Murine NT cells recognize CD1‐expressing cells and are activated in a CD1‐restricted fashion by several synthetic α‐glycosylceramides, such as α‐GalCer. Here we studied the reactivity of human T cells against CD1d+ cells pulsed or not with α‐GalCer and other related ceramides. CD1d‐restricted recognition of α‐GalCer was a general and specific feature of T cell clones expressing both BV11 and canonical AV24AJ18 TCR chains. Besides, human and murine NT cells showed the same reactivity patterns against a set of related glycosylceramides, suggesting a highly conserved mode of recognition of these antigens in humans and rodents. We also identified several AV24BV11 T cell clones self reactive against CD1+ cells of both hemopoietic and nonhemopoietic origin, suggesting the existence of distinct NT cell subsets differing by their ability to recognize self CD1d molecules.

Mechanisms of cell sensitization to a radioimmunotherapy by doxorubicin or paclitaxel in multiple myeloma cell lines

Purpose: The purpose of this study was to analyze different mechanisms (cell cycle synchronization, DNA damage, and apoptosis) that might underlie potential synergy between chemotherapy (paclitaxel or doxorubicin) and radioimmunotherapy with α radionuclides. Experimental Design: Three multiple myeloma cell lines (LP1, RMI 8226, and U266) were treated with 213Bi-radiolabeled B-B4, a monoclonal antibody that recognizes syndecan-1 (CD138) 24 hours after paclitaxel (1 nmol/L) or doxorubicin (10 nmol/L) treatment. Cell survival was assessed using a clonogenic survival assay. Cell cycle modifications were assessed by propidium iodide staining and DNA strand breaks by the comet assay. Level of apoptosis was determined by Apo 2.7 staining. Results: Radiation enhancement ratio showed that paclitaxel and doxorubicin were synergistic with α radioimmunotherapy. After a 24-hour incubation, paclitaxel and doxorubicin arrested all cell lines in the G2-M phase of the cell cycle. Doxorubicin combined with α radioimmunotherapy increased tail DNA in the RPMI 8226 cell line but not the LP1 or U266 cell lines compared with doxorubicin alone or α radioimmunotherapy alone. Neither doxorubicin nor paclitaxel combined with α radioimmunotherapy increased the level of apoptosis induced by either drug alone or α radioimmunotherapy alone. Conclusion: Both cell cycle arrest in the G2-M phase and an increase in DNA double-strand breaks could lead to radiosensitization of cells by doxorubicin or paclitaxel, but apoptosis would not be involved in radiosensitization mechanisms.

The tight interallelic positional coincidence that distinguishes T‐cell receptor Jα usage does not result from homologous chromosomal pairing during VαJα rearrangement

The T‐cell receptor (TCR) α locus is thought to undergo multiple cycles of secondary rearrangements that maximize the generation of αβ T cells. Taking advantage of the nucleotide sequence of the human Vα and Jα segments, we undertook a locus‐wide analysis of TCRα gene rearrangements in human αβ T‐cell clones. In most clones, VαJα rearrangements occurred on both homologous chromosomes and, remarkably, resulted in the use of two neighboring Jα segments. No such interallelic coincidence was found for the position of the two rearranged Vα segments, and there was only a loose correlation between the 5′ or 3′ chromosomal position of the Vα and Jα segments used in a given rearrangement. These observations question the occurrence of extensive rounds of secondary Vα→Jα rearrangements and of a coordinated and polarized usage of the Vα and Jα libraries. Fluorescence in situ hybridization analysis of developing T cells in which TCRα rearrangements are taking place showed that the interallelic positional coincidence in Jα usage cannot be explained by the stable juxtaposition of homologous Jα clusters.

Radiolabeled Antibodies for Cancer Imaging and Therapy

Radiolabeled antibodies were studied first for tumor detection by single-photon imaging, but FDG PET stopped these developments. In the meantime, radiolabeled antibodies were shown to be effective in the treatment of lymphoma. Radiolabeling techniques are well established and radiolabeled antibodies are a clinical and commercial reality that deserves further studies to advance their application in earlier phase of the diseases and to test combination and adjuvant therapies including radiolabeled antibodies in hematological diseases. In solid tumors, more resistant to radiations and less accessible to large molecules such as antibodies, clinical efficacy remains limited. However, radiolabeled antibodies used in minimal or small-size metastatic disease have shown promising clinical efficacy. In the adjuvant setting, ongoing clinical trials show impressive increase in survival in otherwise unmanageable tumors. New technologies are being developed over the years: recombinant antibodies and pretargeting approaches have shown potential in increasing the therapeutic index of radiolabeled antibodies. In several cases, clinical trials have confirmed preclinical studies. Finally, new radionuclides, such as lutetium-177, with better physical properties will further improve the safety of radioimmunotherapy. Alpha particle and Auger electron emitters offer the theoretical possibility to kill isolated tumor cells and microscopic clusters of tumor cells, opening the perspective of killing the last tumor cell, which is the ultimate challenge in cancer therapy. Preliminary preclinical and preliminary clinical results confirm the feasibility of this approach.

213Bi Radioimmunotherapy with an Anti-mCD138 Monoclonal Antibody in a Murine Model of Multiple Myeloma

New multiple myeloma (MM) treatments—such as high-dose melphalan therapy plus autologous stem cell transplantation or regimens incorporating bortezomide, thalidomide, and lenalidomide—substantially increase the rate of complete response that is associated with longer patient survival. Thus, maintaining the complete response status by improving the minimal residual disease after induction therapy is a key goal for MM management. Here, we address the question of radioimmunotherapy efficacy in MM minimal residual disease treatment in mice with a low tumor burden. α-emitters are particularly well adapted to this approach because the short range of α-particles enables localized irradiation of tumor cells within the bone marrow and a cytotoxic effect on isolated cells due to the high LET (linear energy transfer) of α-particles. The CD138 antigen was used as a target because of its strong expression on myeloma cells in 100% of patients. Method: Intravenous injection of 106 5T33 mouse myeloma cells into the Syngeneic mouse strain C57BL/KaLwRij resulted in a rapid invasion of the marrow and limb paralysis, as illustrated by bioluminescence imaging with luciferase-transfected 5T33 cells. Radioimmunotherapy was performed 10 d after 5T33 cell engraftment with an intravenous injection of an antimouse CD138 antibody radiolabeled with 213Bi at activities of 1.85, 3.7, 7.4, and 11.1 MBq. A blood cell count was performed weekly to monitor hematologic toxicity. The levels of blood Flt3 ligand were also measured to evaluate the radioimmunotherapy-related myelotoxicity. Disease progression was monitored by titrating the monoclonal IgG2b antibody produced by 5T33 cells. Results: The groups treated with 3.7 and 7.4 MBq exhibited a median survival greater than 300 and 227 d, respectively, compared with 45.5 d in the control untreated group. The highest activity (11.1 MBq) showed short-term toxicity whereas the lowest activity (1.85 MBq) gave results similar to those of the controls. With activities of 3.7 and 7.4 MBq, mice exhibited a transient hematologic toxicity whereas only temporary and moderate myelotoxicity was observed with 7.4 MBq. Conclusion: This study demonstrates promising therapeutic efficacy of 213Bi-labeled anti-mCD138 for the treatment of residual disease in the case of MM, with only moderate and transient toxicity.

Radioimmunoconjugates for the treatment of cancer.

Radioimmunotherapy (RIT) has been developed for more than 30 years. Two products targeting the CD20 antigen are approved in the treatment of non-Hodgkin B-cell lymphoma (NHBL): iodine 131-tositumomab and yttrium 90-ibritumomab tiuxetan. RIT can be integrated in clinical practice for the treatment of patients with relapsed or refractory follicular lymphoma (FL) or as consolidation after induction chemotherapy. High-dose treatment, RIT in first-line treatment, fractionated RIT, and use of new humanized monoclonal antibodies (MAbs), in particular targeting CD22, showed promising results in NHBL. In other hemopathies, such as multiple myeloma, efficacy has been demonstrated in preclinical studies. In solid tumors, more resistant to radiation and less accessible to large molecules such as MAbs, clinical efficacy remains limited. However, pretargeting methods have shown clinical efficacy. Finally, new beta emitters such as lutetium 177, with better physical properties will further improve the safety of RIT and alpha emitters, such as bismuth 213 or astatine 211, offer the theoretical possibility to eradicate the last microscopic clusters of tumor cells, in the consolidation setting. Personalized treatments, based on quantitative positron emission tomography (PET), pre-therapeutic imaging, and dosimetry procedures, also could be applied to adapt injected activity to each patient.