Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies, II

We have previously described complement-independent killing of human B lymphocytes by two IgM MoAbs derived from the VH4-34 (VH4.21) gene. Analysis of 17 independently derived VH4-34-encoded MoAbs shows that B cell toxicity is not limited to the two described MoAbs, but is a general property shared by a subset of MoAbs derived from the VH4-34 gene. As observed by two independent microscopy techniques, giant membrane pores were formed on target B cells within 10–15 min of exposure to cytotoxic VH4-34-derived MoAbs. Toxicity by individual MoAb correlated directly to its B cell binding intensity measured by FACS, i.e. stronger the binding greater the killing. Sequence analysis showed that V_H region in germ-line or in near germ-line configuration was necessary but not sufficient for B cell binding. In addition, a particular sequence motif enriched in basic amino acids in the CDR3 may be required to supplement the reactivity mediated by the V_H region of the MoAb molecule. VH4-34-encoded antibodies that fulfil the above sequence requirements have cold agglutinin activity towards the i antigen of cord erythrocytes. In vivo, such anti-i/anti-B cell antibodies are rarely detected in healthy adults, but serum levels are dramatically elevated in selective pathological conditions, such as systemic lupus erythematosus and infectious mononucleosis. This strict regulation may be related to the novel and rapid mechanism of human B cell toxicity demonstrated by antibodies encoded by a single human V_H gene.     

Tumour cell killing using chemically engineered antibody constructs specific for tumour cells and the complement inhibitor CD59

Immunotherapy using MoAbs is inefficient due to limited activation of human effectors by mouse antibodies and multiple protective mechanisms available to host cells against autologous complement. We have used chemically engineered antibody constructs and human complement in vitro to specifically target and kill neoplastic B lymphoid cells (Raji). Fab′γFcγ² chimaeric antibody (specific for human CD37) was used to activate the classical pathway of human complement on Raji cells, whilst CD59 was neutralized using one of two different bispecific F(ab′γ)² antibody constructs which contained both cell-targeting (anti-CD19 or anti-CD38) and CD59-neutralizing moieties. When either bispecific construct was used to neutralize CD59, 15–25% of cells were lysed. If CD55 was also neutralized using specific antibody, Raji cells were efficiently killed (70% lysis). When added to a mixture of target (Raji) and bystander (K562) cells, one bispecific antibody (anti-CD38 × anti-CD59) could be specifically delivered to Raji, avoiding significant uptake on CD59-expressing bystander cells (K562). The second bispecific antibody (anti-CD19 × anti-CD59) bound equally well to either cell type. Cell-specific targeting was dependent upon combination of a low-affinity anti-CD59 Fab′γ with a high-affinity anti-tumour cell Fab′γ. When Raji and K562 cells were mixed and incubated with a combination of the engineered constructs and anti-CD55 antibodies, Raji cell lysis (30–40%) was observed in the absence of K562 killing. We propose that combinations of these constructs may be of use for treatments such as ex vivo purging of autologous bone marrow or in vivo targeting of tumour cells.     

Subchronic Effects of Dieldrin and Phenobarbital on Hepatic DNA Synthesis in Mice and Rats

Dieldrin, an organochlorine pesticide, has been shown to behepatocarcinogenic in mice but not rats. Phenobarbital, in contrast,induces hepatic tumors in both mice and rats. Previous studieshave shown that acute dietary exposure of rats or mice to eitherdieldrin or phenobarbital produces several liver changes, includingcentrilobular hypertrophy, induction of hepatic cytochrome P450,and increased liver weight. The present study examined the subchroniceffect of dieldrin (0.1, 1.0, 3.0, 10.0 mg dieldrin/kg diet)and phenobarbital (10, 50, 100, 500 mg phenobarbital/kg diet)on the induction of hepatic DNA synthesis and hepatocyte lethalityin male B6C3F1 mice and male F344 rats. Eight-week-old animalswere treated as above and evaluated for hepatic DNA synthesisafter 7, 14, 21, 28, and 90 days of continual treatment to dieldrinor phenobarbital. Maximal induction of hepatic DNA synthesisin mice was seen at the 14-, 21-, and 28-day sampling times.In rats, no significant increase in hepatic DNA synthesis orhepatocyte lethality was observed at any dose of dieldrin investigated.Phenobarbital produced a significant increase in hepatic DNAsynthesis in both rat and mouse liver following 7 days of treatment.The induction of DNA synthesis in rat liver was transient, withthe labeling index returning to control levels by 14 days oftreatment. In contrast, mice treated with phenobarbital showeda significant increase in hepatic DNA synthesis throughout thetreatment. In both mice and rats, dieldrin and phenobarbitalinduced hepatic DNA synthesis selectively in the centrilobularregion of the hepatic lobule. The lack of an increase in serumenzymes indicative of hepatic damage and the absence of liverhistopathology in mice or rats fed dieldrin or phenobarbitalindicate that the induction of DNA synthesis was not mediatedby a cytolethal, compensatory hyperplastic response, suggestinga mitogenic mechanism. Therefore, the species-specific inductionof hepatic DNA synthesis by either dieldrin or phenobarbitalcorrelated with the previously observed species-specific inductionof hepatic cancer by these two compounds.     

Murine malaria: dissociation of natural killer (NK) cell activity and resistance to Plasmodium chabaudi

Striking differences in the resistance to P. chabaudi infection among different inbred mouse strains have previously been correlated with the level of both the spontaneous and the infection-induced enhanced level of NK cell activity. We have examined this putative correlation in individual animals of backcross progeny derived from A/J (malaria-susceptible, low NK cell activity) and B10.A (malaria-resistant, high NK cell activity) progenitors. We have found that NK cell activity and resistance to malaria segregated independently. Furthermore, C57BL/6-bg/bg mice which are deficient in NK cell activity were found to be as resistant to malaria as their heterozygous C57BL/6-bg/+siblings. We conclude that low NK cell activity, characteristic of A/J strain mice, is not a sufficient determinant of the exquisite susceptibility of these animals to infection with Plasmodium chabaudi.