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The territorial hospital pharmacy (PUI) of the West of Paris, dispenses treatments for a follow-up care instituion (SSR) and a dependent old people's home (EHPAD) via a hospital pharmacy equipped with an automatic machine producing dry oral forms packaged in labeled pillboxes. This project was completed with the support of the Regional Health Agency (ARS) and aims to secure the medication circuit. The methodology for qualifying the computer system and production, the implementation of the medication circuit and the inter-site logistical organization are presented. The prescription software was interfaced with the management software, which is itself interfaced with the robot's, and with the automatic machine's software. Tests and developments allowed to convert dispensing units into prescription units over a period of time. Also, the drugs consumed by these two institutions allowed to establish the therapeutic booklet and drug provision as well as anticipating the automaton filling. Medical devices, solutions and narcotics are exclusively managed as supplies with a specific circuit. Treatments are dispensed weekly (pillboxes and off-robot). For the SSR, an additional daily production concerns the entries of new patients and the modifications of treatment. Automation saves time and security, but human intervention is involved on a daily basis. The institutions have adopted the new organization over about 6 months and the production activity is now smoothed out. In the next few weeks, two other EHPADs with 100 patients will be integrated into the PUI territorial. One of the main challenges is to integrate them into our pre-existing organization, while maintaining their habits.

This study reports the pharmacologic effects of isatuximab, a CD38 mAb, on T- and B-cell acute lymphoblastic leukemia (ALL). We analyzed CD38 expression in 50-T-ALL and 50 B-ALL clinical samples, and 16 T-ALL and 11 B-ALL cell lines. We primarily focused on in vitro assessments of isatuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In vivo assessment of isatuximab activity was performed in several ALL xenograft models, including disseminated and subcutaneous tumor models in female C.B-17 severe combined immunodeficiency mice. Our study reveals that most patients (90%–100%) carried CD38+ blasts independent of disease burden. The median CD38 receptor density on abnormal lymphoblasts is 41,026 copies/cell on T-ALL and 28,137 copies/cell on B-ALL, respectively. In patients with T-ALL, there is a significant increase of CD38 expression in abnormal blasts compared with normal T cells. High-level CD38 receptor density (RD) is critical to trigger effective isatuximab-mediated ADCC against target ALL cells. In addition, a correlation between CD38 RD and isatuximab-mediated ADCP is demonstrated. In the disseminated CD38+, T-ALL, and B-ALL xenograft models, isatuximab is able to induce robust antitumor activity, even at low doses. This study shows that isatuximab has significant in vitro and in vivo activity against ALL cells with robust ADCC and ADCP effects that are associated with CD38 expression levels in both T-ALL and B-ALL.

Immunotoxins and chimeric toxins are hybrid molecules constituted of antibodies, growth factor or cytokines coupled to peptide toxins. They are designed to selectively eliminate tumor cells. Some of these chimera have been shown to induce complete tumor regressions of human tumor xenografts in immunodeficient mice. In clinical trials, higher anti tumor response were observed in lymphoma, brain tumor, breast and colon cancers. Problems arose with normal tissue toxicity and the production of neutralising antibodies. Should the latest recombinant toxins conceived by rationale designed, solved these problems, chimeric toxins would be an alternative approach to target tumor cells and vascular endothelial cells in solid tumors.

Pseudomonas exotoxin (PE) requires proteolytic cleavage to generate a 37-kDa C-terminal fragment that translocates to the cytosol and ADP-ribosylates elongation factor 2. Cleavage within cells is mediated by furin, occurs between arginine 279 and glycine 280, and requires an arginine at both P1 and P4 residues. To study the proteolytic processing of PE-derived chimeric toxins, TGFalpha-PE38 (transforming growth factor fused to the domains II and III of PE) and a mutant form, TGFalpha-PE38gly279, were each produced in Escherichia coli. When assessed on various epidermal growth factor (EGF) receptor-positive cell lines, TGFalpha-PE38 was 100-500-fold more toxic than TGFalpha-PE38gly279. In contrast to PE, where cleavage by furin is only evident at pH 5.5, furin cleaved TGFalpha-PE38 over a broad pH range, while TGFalpha-PE38gly279 was resistant to cleavage. TGFalpha-PE38 was poorly toxic for furin-deficient LoVo cells, unless it was first pretreated in vitro with furin. Furin treatment produced a nicked protein that was 30-fold more toxic than its unnicked counterpart. Using the single chain immunotoxin HB21scFv-PE40 as a substrate, furin-mediated processing of an antibody-based immunotoxin was also evaluated. HB21scFv-PE40, which targets cells expressing the transferrin receptor, was cleaved in a similar fashion to that of TGFalpha-PE38 and nicked HB21scFv-PE40 exhibited increased toxicity for LoVo cells. In short-term experiments, the rate of reduction in protein synthesis by furin-nicked immunotoxins was increased compared with unnicked protein, indicating that cleavage by furin can be a rate-limiting step. We conclude that furin-mediated cleavage of PE-derived immunotoxins is important for their cytotoxic activity.

Recombinant RFB4 single-chain immunotoxin that is cytotoxic towards CD22-positive cells E. Mansfield*, M. F. Chiron*, P. Arnlott, I. Pastan* and D. J. FitzGeraldV *Laboratory of Molecular Biology, DBS, National Cancer Institute, National Institutes of Health, 37/4E I 6, 37 Convent Drive MSC 4255, Bethesda, MS 20892, U.S.A., and tDepartment of Immunology, Royal Free Hospital, Rowland Hill Street, London NW3 2PF, U.K.

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro, furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5-10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli, purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0-5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.