Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.

Novel membrane-bound reporter molecule for sorting high producer cells by flow cytometry.

We developed a membrane bound reporter and selection molecule for sorting by fluorescence activated cell sorting (FACS) of cells producing a protein of interest. This molecule is composed of a transmembrane (TM) domain, fused on its extracellular end to a biotin mimetic peptide (BMP) and on its intracellular side to puromycin N-acetyl transferase (PAC). In this format BMP is displayed on the cell membrane surface and PAC faces the cell cytoplasm. BMP was detected and quantified on the cell surface by fluorescently labelled streptavidin, allowing cell sorting by FACS, according to the reporter expression level. The reporter and a gene of interest (GOI) were connected on the same transcript via an internal ribosomal entry site (IRES). The reporter expression level was found to correlate with that of the GOI, enabling sorting of high producer cells by FACS. Thus, the highest fluorescent cells sorted had also the highest protein of interest (POI) productivity level.

Cytotoxicity of NF-kappaB inhibitors Bay 11-7085 and caffeic acid phenethyl ester to Ramos and other human B-lymphoma cell lines.

OBJECTIVE: The viability of normal and malignant B-cells was shown to depend on the constitutive activation of the nuclear factor (NF)- kappaB pathway. Thus, attempts to find efficient inhibitors of NF-kappaB play a central role in the search for novel anti-B lymphoma therapies. We studied the effects of two NF-kappaB inhibitors, Bay 11-7085 (BAY) and caffeic acid phenethyl ester (CAPE), on the viability of B-lymphoma cell lines. METHODS: We investigated the mechanism(s) of the cytotoxic effect of the NF-kappaB inhibitors, BAY, and CAPE on human-lymphoma and nonhematological cell lines. RESULTS: BAY and CAPE were shown to kill Ramos-Burkitt's lymphoma cells with IC(50) values of 0.7 microM and 4 microM, respectively. The rapid killing by BAY (h) vs the slower killing by CAPE (1-3 days), and their differential effects on the stages of the cell cycle, indicated that these drugs induce killing by different mechanisms. BAY and CAPE induced a loss of the cytoplasmic compartment and generated pyknotic nuclei, which lacked nuclear or nucleosomal fragmentation, features characteristic of necrosis rather than apoptosis. BAY also induced a rapid loss of the mitochondrial potential and rapid inhibition of p65 NF-kappaB binding to its kappaB motif without reducing the level of nuclear p65. CONCLUSION: Our results indicate that BAY causes a necrotic rather than apoptotic cell death, either through its effect on the NF-kappaB pathway and/or by affecting additional molecular targets. The high sensitivity of B-lymphoma cell lines to the cytotoxicity of BAY, justify further research to explore its potential therapeutic effect on human B lymphomas.

Tumor necrosis factor-alpha and CD40L modulate cell surface morphology and induce aggregation in Ramos Burkitt's lymphoma cells.

Interaction of CD40L and its cognate receptor is an essential component of B-lymphocyte signaling, affecting various aspects of B-cell differentiation pathways and immunoglobulin gene expression. However, much less is known about the biological consequences of B-cell signaling through tumor necrosis factor (TNF)-alpha and its cognate receptors TNF-R1 and 2. We used Ramos Burkitt's lymphoma cell line as a model system to study the direct effects of these cytokines on B cells. Treatment of Ramos cells with either TNF-alpha or CD40L, but not with interleukin (IL)- 4, interferon (IFN)-gamma and transforming growth factor (TGF)-beta, resulted in enhanced cell aggregation and enhancement of adherence to glass cover-slips. Scanning electron microscopy showed that Ramos cells have a polarized cell surface morphology and exhibit at least 3 cell surface morphological domains: microvilli, filopodia and ruffled membranes. The cells adhered to the glass matrix through multiple filopodia/podopodia-like cell processes and demonstrated distinct ruffled-like membrane projections on their opposite pole. Induction by TNF-alpha or CD40L, but not with IL-4, IFN-gamma and TGF-beta, resulted in increased number and complexity of both types of membrane projections. TNF-alpha and CD40L upregulated the expression of the adhesion molecule intercellular adhesion molecule-1 and the Fas receptor on Ramos cells, without affecting the expression levels of membrane immunoglobulin M or its secretion rate. Reverse transcriptase-polymerase chain reaction, and flow cytometry demonstrated that Ramos cells expressed TNF-R1 but very little if any TNF-R2, indicating that TNF-alpha exerted its effects on Ramos cells through the former receptor.

Differential effects of tumor necrosis factor-alpha and CD40L on NF-kappa B inhibitory proteins I kappa B alpha, beta and epsilon and on the induction of the Jun amino-terminal kinase pathway in Ramos Burkitt lymphoma cells.

Interaction between the CD40 ligand and its cognate receptor is known to affect various aspects of B-cell biology. Less is known about the biological consequences of B-cell signaling through tumor necrosis factor alpha (TNF-alpha) and its two receptors. We have used Ramos germinal center (GC)-derived Burkitt's lymphoma (BL) cells as a model system to compare some of the early signaling events of TNF-alpha and CD40L on the NF-kappaB and c-Jun amino-terminal kinase (JNK) pathways. We have previously found that both TNF-alpha and CD40L induced enhanced cell aggregation, adherence and modified cell surface morphology of Ramos cells. In the present report, it was found that treatment with either TNF-alpha or CD40L resulted in a rapid degradation (within 15 min) of IkappaBalpha, followed by a recovery period lasting up to a few hours. The level of IkappaBbeta, another inhibitory molecule of the NF-kappaB pathway, also decreased following treatment with CD40L or TNF-alpha. However, whereas CD40L induced a rapid drop without significant recovery within 2 h, TNF-alpha caused a slow and gradual decline of IkappaBbeta. In addition, treatment with CD40L resulted in a gradual and modest decline of up to 60% of the level of IkappaBepsilon within 2 h, whereas a much smaller decline was seen with TNF-alpha (approx. 20%) Our results thus show that in Ramos cells, TNF-alpha and CD40L have common, as well as differential, signaling effects on the IkappaBalpha, IkappaBbeta and IkappaBepsilon, which form inhibitory complex(es) with the NF-kappaB cytosolic proteins. We also found that CD40L, but not TNF-alpha activates the JNK pathway through transient phosphorylation of its threonine183/tyrosine185 residues. As expected, c-Jun, which is known to be a substrate of JNK, was also phosphorylated at serine residue 73 by treatment with CD40L, but not by TNF-alpha.