CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells.

We studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (P<0.001 in both cells) after incubation with CMC-544, but CD20 level remained constant or increased for 12 h. Similar results were obtained in cells from 12 patients with BCM. The antiproliferative and apoptotic effect of CMC-544 were greater than that of rituximab. The ADCC of rituximab was not enhanced by CMC-544. Thus, the combination of CMC-544 and rituximab increased the in vitro cytotoxic effect in BCM cells, and sequential administration for 12 h proceeded by CMC-544 was more effective. The reduction of CD55 and the preservation of CD20 after incubation with CMC-544 support the rationale for the combined use of CMC-544 and rituximab.

A case of coagulation factor V deficiency caused by compound heterozygous mutations in the factor V gene.

We investigated the molecular basis of a severe factor V (FV) deficiency in a Japanese female, and identified two distinct mutations in the FV gene, a novel cytosine insertion (1943insC) and a previously reported point mutation (A5279G). We expected the patient to be a compound heterozygote for those mutations, as a 1943insC, but not an A5279G, was found in the mother and a sibling. The 1943insC will cause a frame-shift after 590Gln, resulting in amino acid substitutions with two abnormal residues followed by a stop codon in the FV A2 domain (FS592X). The A5279G will cause an amino acid alteration in the FV A3 domain (Y1702C), which has been observed in several ethnic groups. We found that both mutant mRNAs were detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the patient's platelets, whereas no FV antigen and activity were detected in plasma. On the one hand, the RT-PCR signal from the FS592X-FV mutant mRNA was markedly reduced, suggesting that the RNA surveillance system would eliminate most of the abnormal FS592X-FV transcripts with a premature termination. On the other hand, expression analyses revealed that only small amounts of Y1702C-FV with a low specific activity were secreted, and that the FS592X-FV was not detected in cultured media. These data indicated that both mutant FV molecules would be impaired, at least in part, during the post-transcriptional process of protein synthesis and/or in secretion. Taken together, it seems to suggest that each gene mutation could be separately responsible for severe FV deficiency, while this phenotype is due to the in-trans combination of the two defects.