Generation of a canine anti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancer patients.

Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a "caninized" version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer.

Identification of the scavenger receptors SREC-I, Cla-1 (SR-BI), and SR-AI as cellular receptors for Tamm-Horsfall protein.

Tamm-Horsfall protein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in urine. An important role for THP in antibacterial host defense but also in inflammatory disorders of the urogenital tract has been suggested. In line with this, THP has been shown recently to potently activate macrophages and dendritic cells (DC) via the toll-like receptor 4 (TLR4) pathway. We show here that THP interacts specifically with surface structures on DC and provides evidence that they are distinct from TLR4. Using retroviral expression cloning, we have identified one such receptor as the scavenger receptor (SR) expressed by endothelial cells I (SREC-I). In addition, we found that two other receptors for acetylated low-density lipoprotein (AcLDL), namely scavenger receptors AI (SR-AI) and Cla-1 (SR-BI), also serve as receptors for THP. SREC-I/THP interaction is of high affinity (16.8+/-6.8 nM), whereas Cla-1 and SR-AI have lower affinities for THP (396 nM+/-114 nM and 802 nM+/-157 nM, respectively). The interaction of THP with these molecules is fully blocked by AcLDL. However, AcLDL only partially blocks binding of THP to DC, and a series of experiments did not support a role in DC activation for SR interacting with THP and AcLDL. Thus, our data point to the existence of additional receptors for THP, which mediate TLR4-dependent DC activation. Interaction and up-take of THP by SR might play an important role in local host defense and could contribute to inflammatory kidney diseases associated with THP-specific antibody responses.

Sustained expression of early growth response protein-1 blocks angiogenesis and tumor growth.

Transient induction of the transcription factor early growth response protein-1 (EGR-1) plays a pivotal role in the transcriptional response of endothelial cells to the angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which are produced by most tumors and are involved in the angiogenic switch. We report here that sustained expression of EGR-1 by recombinant adenoviruses in endothelial cells, however, leads to the specific induction of potent feedback inhibitory mechanisms, including strong up-regulation of transcriptional repressors, negative cell cycle check point effectors, proteins with established antiangiogenic activity, and several proapoptotic genes. Sustained EGR-1 expression consistently leads to an antiangiogenic state characterized by an altered responsiveness to VEGF and bFGF and a striking inhibition of sprouting and tubule formation in vitro. Furthermore, EGR-1-expressing viruses potently inhibit cell invasion and vessel formation in the murine Matrigel model and repress tumor growth in a murine fibrosarcoma model. We propose that gene therapy involving sustained EGR-1 expression may constitute a novel therapeutic principle in the treatment of cancer due to the simultaneous induction of multiple pathways of antiangiogenesis, growth arrest, and apoptosis induction in proliferating cells leading to preferential inhibition of angiogenesis and tumor growth.

Tamm-Horsfall glycoprotein links innate immune cell activation with adaptive immunity via a Toll-like receptor-4-dependent mechanism.

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-kappaB. Bone marrow-derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF-alpha production was evident in WT but not in Tlr4-/- mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4-/- mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.

Differential expression of inhibitory and activating CD94/NKG2 receptors on NK cell clones.

Natural killer cells are known to express a variety of surface receptors involved in HLA class I monitoring. It is thus of interest to investigate the clonal distribution and relative expression levels of activating versus inhibitory NK receptors. We have developed a quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay designed to determine specific and absolute mRNA levels for NKG2-A/B, -C, -E, -F, -H and NKG2-D. When analyzing NK cell clones derived from a single donor we found differential expression of inhibitory (NKG2-A/B) versus triggering (NKG2-C and potentially -E, -F, -H) NK receptor chains. The generation of the splice variants NKG2-E and -H seemed to occur at a constant ratio. We further compared NKG2 transcript levels to surface receptor expression as monitored by flow cytometric analysis and to NK cell cytotoxicity as detected by reverse ADCC: a clear correlation was observed. Thus, the data obtained reveal a substantial variability in the NKG2 repertoire among NK cell subpopulations, which is likely to affect the sensitivity and reactivity towards the ligand HLA-E.