The autophilic anti-CD20 antibody DXL625 displays enhanced potency due to lipid raft-dependent induction of apoptosis.

Despite widespread use of anti-CD20 antibodies as therapeutic agents for oncologic and autoimmune indications, precise descriptions of killing mechanisms remain incomplete. Complement-dependent cytolysis and antibody-dependent cell-mediated cytotoxicity are indicated as modes of target cell depletion; however, the importance of apoptosis induction is controversial. Studies showing that the therapeutic anti-CD20 antibody rituximab (Rituxan) mediates apoptosis of tumor cell targets in vitro after cross-linking by anti-Fc reagents suggest that enhancement strategies applied to Fc-independent activities for anti-CD20 antibodies could improve therapeutic efficacy. An anti-CD20 antibody designated DXL625, with autophilic properties such as increased binding avidity, is shown here to independently induce caspase-mediated apoptosis of an established B-cell lymphoma line in vitro. Depletion of membrane cholesterol or chelation of extracellular calcium abrogated the pro-apoptotic activity of DXL625, indicating that intact lipid rafts and calcium are required for this activity. The Fc-mediated complement-dependent and antibody-dependent cellular killing mechanisms are maintained by DXL625 despite conjugation of the parental Rituxan antibody to the autophilic DXL peptide sequence. This study shows a strategy for improving anti-CD20 immunotherapy by endowing therapeutic antibodies with self-interacting properties.

Quantification of HTLV-I proviral load in experimentally infected rabbits.

BACKGROUND: Levels of proviral load in HTLV-1 infected patients correlate with clinical outcome and are reasonably prognostic. Adaptation of proviral load measurement techniques is examined here for use in an experimental rabbit model of HTLV-1 infection. Initial efforts sought to correlate proviral load with route and dose of inoculation and with clinical outcome in this model. These methods contribute to our continuing goal of using the model to test treatments that alleviate virus infection. RESULTS: A real-time PCR assay was used to measure proviral load in blood and tissue samples from a series of rabbits infected using HTLV-1 inocula prepared as either cell-free virus particles, infected cells or blood, or by naked DNA injection. Proviral loads from asymptomatically infected rabbits showed levels corresponding to those reported for human patients with clinically silent HTLV-1 infections. Proviral load was comparably increased in 50% of experimentally infected rabbits that developed either spontaneous benign or malignant tumors while infected. Similarly elevated provirus was found in organs of rabbits with experimentally induced acute leukemia/lymphoma-like disease. Levels of provirus in organs taken at necropsy varied widely suggesting that reservoirs of infections exist in non-lymphoid organs not traditionally thought to be targets for HTLV-1. CONCLUSION: Proviral load measurement is a valuable enhancement to the rabbit model for HTLV-1 infection providing a metric to monitor clinical status of the infected animals as well as a means for the testing of treatment to combat infection. In some cases proviral load in blood did not reflect organ proviral levels, revealing a limitation of this method for monitoring health status of HTLV-1 infected individuals.

Binding of HTLV-1 virions to T cells occurs by a temperature and calcium-dependent process and is blocked by certain type 2 adenosine receptor antagonists.

A flow cytometric assay that measures binding of human T-lymphotropic virus type 1 (HTLV-1) virions to target cells was used to investigate the binding process and to screen for compounds affecting viral binding. Results showed that adenosine receptor type 2 antagonists effectively inhibit viral binding at concentrations below 10 microM; no inhibition was seen when antagonist was used to pretreat cells or was added post binding, suggesting direct interference with virus attachment. Efficient HTLV-1 binding required divalent calcium ions and temperatures greater than 20 degrees C. Disruption of lipid rafts by cholesterol depletion compromised viral binding but cleavage of glycosyl-phosphatidylinositol linkages had no effect. A monoclonal antibody (mAb) that recognizes HTLV-1 envelope positions 190-209 impaired binding of virus while other anti-envelope antibodies had no effect. These findings place major constraints on the nature of the HTLV-1 cell binding process and identify a class of inhibitors that may have potential for treatment of infection.

Monoclonally integrated HTLV type 1 in epithelial cancers from rabbits infected with an HTLV type 1 molecular clone.

In addition to T cell leukemias and lymphomas, human T cell leukemia virus type 1 (HTLV-1) infection has been associated with nonhematologic malignancies and described as the cause of one case of small-cell lung carcinoma. Infected primary epithelial cells have been isolated from sweat gland and oral mucosae of HTLV-1-infected human patients. In the present study, epithelial neoplasms developed in two rabbits experimentally infected with a molecular clone of HTLV-1 (strain K30p). Serologic detection of anti-HTLV-1 and isolation of virus from blood lymphocytes at multiple time points postinjection established a course of chronic asymptomatic infection in both. One rabbit, infected for 5.5 years after intramuscular injection of HTLV-1 DNA, developed a thymoma having features of medullary differentiation. HTLV-1 provirus was detected in both thymocytes and neoplastic epithelium isolated discretely from the thymoma by laser capture microdissection. These findings provide the first experimental evidence of HTLV-1 disease after infection by HTLV-1 DNA injection. Endometrial adenocarcinoma occurred in a second rabbit 2.5 years after its inoculation with cell-associated virus. In this second case, an epithelial cell line derived ex vivo from a metastatic lesion produced virus in culture. In tumors from each of the two rabbits, the neoplastic epithelium was infected and harbored monoclonally integrated HTLV-1 provirus. Although monoclonal provirus integration alone does not establish retroviral cause of carcinogenesis unequivocally, these and other accumulating data indicate that there may be a role for HTLV-1 in diseases associated with infection of epithelia, including some epithelial cancers.

Gene expression in mice infected with West Nile virus strains of different neurovirulence.

West Nile virus causes febrile illness in humans with a proportion of cases progressing to meningoencephalitis, encephalitis, hepatitis, and death. Isolates of the virus fall into two genetic lineages, with differences in neuroinvasiveness for mice occurring between strains within both lineages. We used DNA microarrays to compare gene expression in mice infected peripherally with seven lineage 1 and 2 strains confirmed to be of either high or low neuroinvasiveness in mice and associated with severe or benign infection in humans and birds. The 4 strains with highest neuroinvasiveness induced increased expression of 47 genes in the brain, 111 genes in the liver, and 70 genes in the spleen, relative to the 3 least neuroinvasive strains. Genes involved in interferon signaling pathways, protein degradation, T-cell recruitment, MHC class I and II antigen presentation, and apoptosis were identified that may have both pathogenic and protective effects, but increased expression of certain acute proteins, central nervous system specific proteins and proteins associated with T-cell hepatitis, implicate mechanisms related to exalted virulence.