Differential regulation of B-cell proliferation by IL21 in different subsets of chronic lymphocytic leukemia.

Interleukin-21 (IL21) plays an important role in B-cell proliferation, survival and differentiation. Contrary to its stimulatory effect in normal B cells, it has been shown that it induces pro-apoptotic effect in leukemic B cells from CLL patients. Little is known regarding the biological function of IL21 in leukemic B cells from progressive and non-progressive CLL patients. In the present study, the proliferative effect of IL21 in combination with TLR9 agonist (CpG) was investigated in B cells isolated from 24 CLL patients and eight normal subjects by radioactive thymidine incorporation assay. B cells were enriched from peripheral blood mononuclear cells by negative selection using magnetic beads (MACS) and immunophenotyped by flow cytometry. Our results showed that IL21 enhanced the proliferative effects of CpG in both normal and leukemic B cells, though no significant differences were observed between CLL patients and healthy controls. Comparison between different subsets of patients revealed that while the combination of IL21 and CpG significantly inhibited the proliferation of B cells from progressive compared to non-progressive patients (p=0.001), it enhanced proliferation of leukemic B cells from IGHV mutated compared to unmutated patients (p=0.001). The inhibitory effect of IL21 on proliferation of normal and leukemic cells was found to be apoptosis-independent. Our findings suggest differential effects of IL21 in different subsets of CLL patients and suggest its potential therapeutic implication in patients with a more progressive disease.

Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C.

Clostridium tetani causes a life-threatening infectious disease by production of tetanus neurotoxin (TeNT), a 150 kDa molecule composed of light (LC) and heavy chain (HC) polypeptides. The TeNT HC contains an N-terminal domain critical for LC translocation and a C-terminal toxin receptor-binding domain known as fragment C. Despite extensive investigations on epitope specificity of anti-TeNT antibodies, the immunodominant neutralizing epitopes of the toxin are poorly defined. This study describes the generation and characterization of four monoclonal antibodies (MAb) specific for TeNT. The characteristics of each MAb were explored in terms of isotype, specificity, affinity, and immuno-globulin heavy chain variable region (IGHV) gene usage using ELISA, Western blotting, and sequencing techniques. The toxin neutralizing activity of the MAbs was also investigated using the in vitro GT1b neutralizing assay. The data demonstrated that all MAbs bind to tetanus toxin and toxoid. Sub-fragments binding analysis showed that two MAbs react with fragment C, one with both fragment C and LC, and one with LC. Only the two fragment C-specific MAbs were able to neutralize the toxin. Sequencing of the expressed VH and VL genes revealed rearrangements of various VH and VL gene segments in all hybridoma clones. Clonality of the hybridomas was also confirmed by a competition assay that showed recognition of distinct epitopes by these MAbs. The results suggest the importance of TeNT fragment C in terms of immunogenicity and toxin neutralization activity.

In vitro assessment of the effects of anti-HER2 monoclonal antibodies on proliferation of HER2-overexpressing breast cancer cells.

BACKGROUND: HER2 proto-oncogene is critical in the biology of breast cancer and an important therapeutic target of monoclonal antibodies (mAbs). We have recently established a panel of anti-HER2 mAbs recognizing different epitopes within the extracellular domain of HER2. MATERIALS & METHODS: In the present study the antiproliferative effect of these mAbs was investigated on HER2-overexpressing human breast cancer cell line BT474, using radioactive thymidine incorporation assay. RESULTS: Our results demonstrated that while two of the mAbs (1T0 and 2A8) inhibited cell proliferation dose dependently, similar to trastuzumab, six mAbs (1F2, 1B5, 1H9, 4C7, 1H6 and 2A9) augmented cell proliferation. Treatment of BT474 cells with different combinations of two mAbs induced either synergistic inhibitory or stimulatory effects. DISCUSSION: Our findings indicate that combination of some stimulatory mAbs could completely abolish the inhibitory effect of other mAbs against HER2. Employment of some combinations of mAbs with significant synergistic inhibitory function may improve the therapeutic efficacy of HER2-specific mAbs.

Optimization of an anti-HER2 monoclonal antibody targeted delivery system using PEGylated human serum albumin nanoparticles.

Human serum albumin (HSA) nanoparticles represent an attractive strategy for active targeting of therapeutics into tumor cells due to the presence of superficial functional groups. HER2 is highly expressed in a significant proportion of cancers and monoclonal antibodies (mAbs) directed against HER2 hold great promise for effective therapy. Herein, covalent coupling of a novel mAb (1F2) directed against the extracellular domain of HER2 to the surface of HSA nanoparticles was evaluated to obtain nanoparticles with highest cellular uptake. HER2 reactivity of 1F2-conjugated nanoparticles produced under different conditions was screened by an indirect ELISA and flow cytometry techniques. Monoclonal antibody thiolation with 100-fold molar excess of 2-iminothiolane and the ratio of 10:1 for the thiolated 1F2 (µg) to PEGylated nanoparticles (mg), were optimum for the attachment process. Under this condition, 23±4% of 1F2 was conjugated to nanoparticles. The flow cytometry results show that 1F2-modified nanoparticles interact with nearly all HER2 receptors on the surface of BT474 cells. In addition, no cellular uptake was observed on MCF7 cells. In vitro analyses showed no significant cytotoxicity of produced system against BT474 cells. Therefore, 1F2-attached HSA nanoparticles represent a potential delivery system for targeted transport of therapeutic agents into HER2-positive tumor cells.

Characterization of novel murine monoclonal antibodies directed against the extracellular domain of human HER2 tyrosine kinase receptor.

HER2 proto-oncogene encodes a transmembrane receptor tyrosine kinase overexpressed in a variety of solid tumors. Several mouse monoclonal antibodies (MAbs) have been developed that recognize the extracellular part of HER2; of them two MAbs were humanized and employed for targeted immunotherapy. In this study we aimed to produce murine MAbs that specifically recognize the extracellular domain of human HER2. BALB/c mice were first primed with HER2-transfected NIH-3T3 cells and then boosted with recombinant extracellular part of HER2. Splenocytes from hyperimmunized mice were fused with myeloma cells and growing hybridomas were selected and screened for HER2 reactivity by an indirect ELISA. HER2-specific hybridomas were selected, cloned by limiting dilution assay, and further characterized by Western blotting and flow cytometry techniques. All clones showed positive reactivity to HER2 with binding affinity, ranging from 1.9×10(8) to 5×10(9), and stained HER2-transfected cells and malignant cells overexpressing HER2. None of the MAbs inhibited the binding of trastuzumab (Herceptin(®)) to HER2, indicating recognition of distinct epitopes by these MAbs. Based on these findings, our MAbs could be potentially used for selective targeting of HER2-expressing malignancies.