Adcitmer® , a new CD56-targeting monomethyl auristatin E-conjugated antibody, is a potential therapeutic approach in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer, whose tumour cells often express CD56. While immune checkpoint inhibitors constitute a major advance for treating patients with MCC with advanced disease, new therapeutic options are still urgently required. OBJECTIVES: To produce and evaluate the therapeutic performance of a new antibody-drug conjugate (Adcitmer® ) targeting CD56 in preclinical models of MCC. METHODS: CD56 expression was evaluated in a MCC cohort (immunohistochemistry on a tissue microarray of 90 tumour samples) and MCC cell lines. Interaction of an unconjugated CD56-targeting antibody with CD56+ MCC cell lines was investigated by immunohistochemistry and imaging flow cytometry. Adcitmer® product was generated by the bioconjugation of CD56-targeting antibody to a cytotoxic drug (monomethyl auristatin E) using the McSAF Inside® bioconjugation process. The chemical properties and homogeneity of Adcitmer® were characterized by hydrophobic interaction chromatography. Adcitmer® cytotoxicity was evaluated in vitro and in an MCC xenograft mice model. RESULTS: Similar to previous reports, CD56 was expressed by 66% of MCC tumours in our cohort, confirming its relevance as a therapeutic target. Specific binding and internalization of the unconjugated CD56-targeting antibody was validated in MCC cell lines. The high homogeneity of the newly generated Adcitmer® was confirmed by hydrophobic interaction chromatography. The CD56-mediated cytotoxicity of Adcitmer® was demonstrated in vitro in MCC cell lines. Moreover, Adcitmer® significantly reduced tumour growth in a MCC mouse model. CONCLUSIONS: Our study suggests that Adcitmer® should be further assessed as a therapeutic option in patients with MCC, as an alternative therapy or combined with immune checkpoint inhibitors.

Microdosimetry in low energy proton beam at therapeutic-equivalent fluence rate with silicon 3D-cylindrical microdetectors.

In this work we show the first microdosimetry measurements on a low energy proton beam with therapeutic-equivalent fluence rates by using the second generation of 3D-cylindrical microdetectors. The sensors belong to an improved version of a novel silicon-based 3D-microdetector design with electrodes etched inside silicon, which were manufactured at the National Microelectronics Centre (IMB-CNM, CSIC) in Spain. A new microtechnology has been employed using quasi-toroid electrodes of 25µm diameter and a depth of 20µm within the silicon bulk, resulting in a well-defined cylindrical radiation sensitive volume. These detectors were tested at the 18 MeV proton beamline of the cyclotron at the National Accelerator Centre (CNA, Spain). They were assembled into an in-house low-noise readout electronics to assess their performance at a therapeutic-equivalent fluence rate. Microdosimetry spectra of lineal energy were recorded at several proton energies starting from 18 MeV by adding 50µm thick tungsten foils gradually at the exit-window of the cyclotron external beamline, which corresponds to different depths along the Bragg curve. The experimentalyF¯values in silicon cover from (5.7 ± 0.9) to (8.5 ± 0.4) keV µm-1in the entrance to (27.4 ± 2.3) keV µm-1in the distal edge. Pulse height energy spectra were crosschecked with Monte Carlo simulations and an excellent agreement was obtained. This work demonstrates the capability of the second generation 3D-microdetectors to assess accurate microdosimetric distributions at fluence rates as high as those used in clinical centers in proton therapy.

SV40 induced cellular immortalization: phenotypic changes associated with the loss of proliferative capacity in a conditionally immortalized cell line.

Immortalization of rodent embryo fibroblasts by SV40 is dominantly maintained by the large T antigen. The aim of this work is to characterize some of the events associated with the loss of proliferative capacity in a rat cell line, called REtsAF, which is conditionally immortalized by the tsA58 allele of SV40 large T antigen. DNA replication is arrested less than 24 h after the shift to the restrictive temperature (39 degrees C). This arrest occurs without specificity relative to the cell cycle stage, which suggests that a function essential throughout the cell cycle is affected. A two-dimensional SDS polyacrylamide gel electrophoresis analysis of proteins shows that, although the global rate of protein synthesis is only slightly affected at 39 degrees C, the rate of accumulation of specific proteins is either increased or decreased. Finally we present biochemical and electron microscopy data showing that alterations of the mitochondria occur upon shift to 39 degrees C.

Recognition by the DNA repair system of DNA structural alterations induced by reversible drug-DNA interactions.

Ditercalinium (NSC 335153) was synthesized as a bifunctional DNA intercalator. It is made of two 7-H pyridocarbazole rings joined by a rigid bis-ethyl bispiperidine chain. It binds to DNA with high affinity and elicits anti-tumor activity on a variety of animal tumors. 1H n.m.r. studies of ditercalinium bis-intercalated into d(CpGpCpG)2 have shown that the intercalation process occurs from the large groove of the DNA helix while the two intercalated rings are separated by two base pairs. Because of the linking chain rigidity of ditercalinium, DNA conformation has to be altered to permit the intercalation of the two rings. DNA must be bent toward the minor groove. In E. coli, ditercalinium elicits a specific toxicity on polA strains which is suppressed by an additional uvrA mutation. In vitro, the purified UvrA and UvrB proteins bind to the DNA-ditercalinium complex in an ATP dependent manner. The UvrABC complex induces single-strand nicks, but only when ditercalinium is bound to negatively supercoiled DNA. The life-time of the UvrAB-DNA-ditercalinium complex is greater than 50 min when free ditercalinium concentration is maintained constant in the incubation medium. The cytotoxicity of ditercalinium in E. coli results from the induction of a futile and abortive DNA repair. The reversible ditercalinium-DNA complex mimics a bulky DNA lesion, yet the UvrABC endonuclease is unable to cope with a reversible lesion since it cannot eliminate the causative agent. The interaction of UvrA and UvrB proteins has also been studied with DNA and other DNA-binding drugs forming high-affinity complexes such as distamycin. The Uvr protein recognition process appears to be associated with specific DNA structural alterations. In eukaryotic cells, ditercalinium is concentrated in mitochondria. Mitochondrial DNA is rapidly and totally degraded. Mitochondrial DNA coded proteins being no longer synthesized, the respiratory chain is progressively inactivated. The stimulation of the glycolytic pathway allows the cells to continue growth for several generations. Dihydro-orotate dehydrogenase is located in the inner membrane of mitochondria and its activity is dependent on mitochondria energization. It becomes inactive after ditercalinium treatment. A drop of the pyrimidine pool is then observed. Complementation of treated cells with uridine decreases 10-fold the ditercalinium toxicity. The cellular delayed toxicity of ditercalinium results from the slow induction of a pyrimidineless state associated with the progressive inactivation of mitochondria. The results show that DNA structural alterations induced by reversible drug-DNA complexes can be recognized by DNA repair enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective alteration of mitochondrial function by Ditercalinium (NSC 335153), a DNA bisintercalating agent.

The bifunctional intercalator Ditercalinium (NSC 335153) demonstrates an anti-tumoral cytotoxicity markedly different from other intercalating agents. A delayed toxicity is observed in eucaryotic cells, both in vitro and in vivo, at drug concentrations far below those required to observe immediate toxic effects. Fluorescence microscopy demonstrates that Ditercalinium and the mitochondrial-staining fluorophore DiOC2(5) are concentrated in the same cellular organelles of L1210 cells. Electron microscopy of Ditercalinium-treated cells reveals extensive and progressive swelling of mitochondria, with no other ultrastructural changes observed. Ditercalinium uptake and toxicity are in part related to mitochondrial membrane potential. However, drug accumulation itself does not immediately alter the mitochondrial membrane potential. Cellular ATP pool levels and the rate of respiration fall progressively after drug treatment. Nucleotide pools in DC3F cells, measured between drug treatment and death, show marked drops in pyrimidine levels while purine nucleotide levels decline more slowly. Addition of uridine or cytidine partially rescues Ditercalinium-treated cells, while toxicity is increased in the presence of 2-deoxyglucose. The combined evidence indicates that the toxicity of Ditercalinium to murine leukemia cells (L1210) and Chinese Hamster lung cells (DC3F) is due to disruption of mitochondrial function.

Rational design of bis-intercalating drugs as antitumour agents: importance of rigidity in the linking chain.

Ditercalinium (NSC 366241), a dimer of 10-methoxy-7H-pyrido[4,3-c]carbazole quaternarized on the pyridine nitrogen by a rigid bis(1,1'-ethyl)-4,4'-bipiperidine linking chain, is endowed with antitumour properties and bis-intercalates with high affinity into DNA. New dimers have been designed in the same series to evaluate the importance of the rigidity of the linking chain for pharmacological activity. The dimers, characterized by one and two additional methylene groups between the two piperidine rings of the linking chain, remain as active as ditercalinium. However, a third additional CH2 group between the two piperidine rings leads to an inactive dimer. Relationships between the different pharmacological activities of the drugs and their intercalation complexes with DNA were investigated using viscosimetry, absorption spectroscopy and NMR analyses.

Effects of the bifunctional antitumor intercalator ditercalinium on DNA in mouse leukemia L1210 cells and DNA topoisomerase II.

Ditercalinium, a 7H-pyridocarbazole dimer (bisintercalator) belongs to a new class of antineoplastic intercalating agents. To investigate its mechanism of cytotoxicity, the effects of ditercalinium on DNA were assessed using normal (L1210) and drug-resistant (L1210/PyDi1) mouse leukemia cells. Alkaline elution assays demonstrated that ditercalinium produced no DNA strand breaks, DNA-protein cross-links, or DNA-DNA cross-links, eliminating these effects as cytotoxic lesions. This result sets ditercalinium apart from other intercalating agents with respect to its interaction with DNA. Nucleoids (histone-depleted chromatin) from ditercalinium-treated L1210 cells were considerably more compact than those from untreated cells, as determined by sedimentation in neutral sucrose gradients. In contrast, nucleoids from ditercalinium-treated L1210/PyDi1 (resistant) cells were similar in compactness to those from control cells. Thus, ditercalinium altered chromatin structure in vivo. The effect of the bisintercalator on purified DNA topoisomerase II, an intracellular target of monointercalators, was measured in vitro. Ditercalinium (5 X 10(-7) M) completely inhibited both the formation of covalent complexes between this enzyme and simian virus 40 DNA and the enzyme-induced DNA cleavage. In addition, ditercalinium induced DNA catenation in the presence of topoisomerase II and adenosine triphosphate. Thus, the cytotoxicity of ditercalinium may derive from a mechanism that, although involving topoisomerase II, is manifested by condensation of DNA rather than by the induction of protein-associated DNA strand breaks.

Effects of new antitumor bifunctional intercalators derived from 7H-pyridocarbazole on sensitive and resistant L 1210 cells.

The antitumor properties of 7H-pyridocarbazole dimers, a new series of bifunctional intercalators, have recently been described (Pelaprat, D. Delbarre, A., Le Guen, I., Roques, B. P., and Le Pecq, J. B. J. Med. Chem., 23: 1336-1343, 1980; and Roques, B. P., Pelaprat, D., Le Guen, I., Porcher, G., Gosse, C., and Le Pecq, J. B. Biochem. Pharmacol., 28: 1811-1815, 1979). In order to study the mechanism of action of these compounds, an L1210 subline was made resistant to one dimer (NSC 335153; ditercalinium). Selection of resistant cells was based on an in vitro-in vivo procedure as follows. Ascitic cells were taken from a leukemic mouse and incubated in vitro with the dimer for 1 hr. They were then injected into mice. After the development of the ascites, L1210 cells were collected and the process was repeated 13 times, until establishment of the resistance. Cloned resistant cells have maintained their resistance for 18 months of in vitro culture. The effects of two dimers (NSC 335153 and NSC 335154) on cell viability, growth, colony formation, and cell cycle progression were investigated on parental and resistant L1210 cells. The cross-resistance of these two L1210 cell lines to several cytotoxic agents was estimated. Several observations indicate that the mechanism of action of these dimers might be different from that of monointercalating agents: (a) these drugs induce a delayed toxicity (growth arrest occurring five generations after drug exposure) in sensitive but not in resistant cells; (b) cells exposed to the dimers arrested almost randomly in all phases of the cell cycle, whereas the corresponding monomer provokes a block in the G2 + M phase. Resistant cells were cross-resistant to 7H-pyridocarbazole monomer, Adriamycin, and vincristine but not to 6H-pyridocarbazole monomer derivatives, actinomycin D, and methotrexate.

DNA and protein changes in the spermatozoa of bulls treated orally with ethylene dibromide.

Oral treatment of bulls with ethylene dibromide caused a temporary reduction of the DNA and protein content and head area of epididymal and ejaculated spermatozoa.

[Relative concentration of deoxyribonucleic acid in the experimental deciduoma and the blastocyst of female rats: cytophotometric study]. / Teneur relative en acide désoxyribonucléique dans le deciduome expérimental et le blastocyste chez la ratte: étude cytophotometrique

Polyploidy level of experimentally induced deciduomata was determined by cytophotometric measures of DNA-Feulgen, with the "Ultramikrospektrophotometer UMSP Zeiss" by scanning. The antimesometrial tissue reached 32 n, 72 H after the uterine trauma, whereas the mesometrial part did not exceed a 4 n polyploidy level. With the same technics we studied the ovarian hormone, its action upon the blastocyst cellular cycle. In delayed blastocysts, synthesis was never found. OEstradiol removed the inhibition and DNA synthesis then starts again.

[Cytophotometric determination of the relative amounts of deoxyribonucleic acid in the nuclei of blastocysts during normal and retarded implantation in the rat].

Cytophotometrics measures of DNA-Feulgen were realized in normal and delayed implantation blastocysts of the rat. During afternoon of the 5th day of progestation, blastocysts were of two types: those nuclei of which were in G1 gap and those nuclei of which were synthetising DNA. In delayed implantation, blastocyst nuclei were in G1 or G2 gap. Mitosis were rarely seen. After oestrogen injection DNA-synthesis started again. Cell kinetics before ovoimplantation is discussed.

[Cytophotometric study of deoxyribonucleic acid in mesometral cells of experimental deciduoma in unilaterally pregnant rats]. / Etude cytophotométrique de l'acide désoxyribonucléique dans les cellules mésométrales du déciduome expérimental chez la ratte en grossesse unilatérale

Ploidy level of mesometral cells in experimentaly induced deciduomata is determinated by DNA-Feulgen cytophotometric measures. This tissue shown the first evolutive stages leading to polyploïdy (mono-binucleated cells) it was unable to reach a ploïdy level higher than 4 n.