Development of DS-5573a: A novel afucosylated mAb directed at B7-H3 with potent antitumor activity.

B7-H3 is highly overexpressed in a variety of human clinical tumors, and its expression is significantly associated with poor outcomes. In our study, we aimed to develop new antitumor mAbs by employing cancer cell immunization, and succeeded in generating a mouse anti-human B7-H3 antibody (M30) that shows antitumor activity. M30 was humanized (Hu-M30), and an afucosylated Hu-M30 (DS-5573a) was also generated. To assess the potency of DS-5573a as a therapeutic mAb, we characterized this mAb and evaluated its antitumor activity in vitro and in vivo. Flow cytometry analysis showed that B7-H3 proteins were expressed on various types of cancer cell lines broadly, and DS-5573a binds to IgC1 and IgC2 domains of human B7-H3. Antibody-dependent cellular cytotoxicity activity of DS-5573a was drastically enhanced against medium to high B7-H3-expressing cancer cell lines MDA-MB-231 and NCI-H322. DS-5573a also induced high antibody-dependent cellular cytotoxicity activity against low B7-H3-expressing cancer cell line COLO205, whereas Hu-M30 induced little activity against it. In addition, DS-5573a was found to be a novel anti-B7-H3 antibody which showed antibody-dependent cellular phagocytosis activity. Furthermore, DS-5573a showed dose-dependent and significant antitumor efficacy (0.03-3 mg/kg) in MDA-MB-231-bearing SCID mice (which have functional natural killer cells and macrophages), but little antitumor efficacy in NOG mice (which lack natural killer cells and have reduced macrophage function). These results suggest that antitumor activity of DS-5573a is mediated by effector cells, and this mAb could be a promising antitumor therapy for patients with a wide range of B7-H3-expressing tumors.

Anti-tumor effect of adenoviral vector-mediated p53 gene transfer on the growth of canine osteosarcoma xenografts in nude mice.

We evaluated the anti-tumor effect of adenoviral vector-mediated p53 gene therapy on the growth of canine osteosarcoma xenografts formed in nude mice. Nude mice were subcutaneously transplanted with cells of 2 P53 mutant canine osteosarcoma cell lines, POS and CHOS. The osteosarcoma xenografts were injected with either an adenoviral vector that expresses canine wild-type P53 (AxCA-cp53) or LacZ (AxCA-LacZ). Tumor growth was significantly inhibited in the xenografts injected with AxCA-cp53 in comparison to those injected with AxCA-LacZ or PBS during the observation period of 27 days. An increase of the amount of p21(WAF1/CDKN1A) mRNA, and the number of apoptotic cells was shown in the tumors injected with AxCA-cp53 in comparison to those injected with AxCA-LacZ or PBS. The present study revealed that the adenoviral vector-mediated p53 gene transfer had an anti-tumor effect in canine osteosarcoma xenografts formed in nude mice.

Establishment and characterization of eight feline mammary adenocarcinoma cell lines.

Eight new feline mammary adenocarcinoma cell lines derived from either primary or metastatic lesions were established. The morphology of all the cell lines was epithelioid and round to spindle in shape, with cell growth occurring in a monolayer fashion. On immunohistochemistry, these cells reacted with anti-keratin and anti-vimentin antisera. The doubling time of these cells was between 19 and 54 hr. Tumor masses were developed in nude mice by subcutaneous inoculation of the cells that were histologically identical to their original mammary tumor lesions. Telomerase activities measured using the telomeric repeat amplification protocol assay revealed high telemetric activity in all of the cells.

High expression of Bcl-xL in delayed apoptosis of canine neutrophils induced by lipopolysaccharide.

The expression of Bcl-2 family members, Bcl-xL, Bcl-2, Mcl-1 and Bax was investigated in delayed apoptosis of canine neutrophils induced by lipopolysaccharide (LPS). Apoptotic cell rates in neutrophils stimulated by LPS (100 ng/ml) were measured at 24 h incubation by TUNEL assay. The incidence of apoptotic neutrophils stimulated by LPS at 24 h incubation was 17.0+/-2% and that in non-stimulated neutrophils was 29.9+/-3%. By real-time quantitative PCR analysis, it was indicated that Bcl-xL and Bax levels in canine neutrophils were significantly affected by LPS stimulation. The levels of Bcl-xL, Bcl-2, Mcl-1 and Bax transcripts at 9 h incubation in neutrophils stimulated by LPS (100 ng/ml) were increased by about 80.4-, 1.9-, 1.4- and 5.3-folds, in comparison to those in non-stimulated neutrophils, respectively. These results indicated that Bcl-xL was proved have an important role in the inhibition of canine neutrophil apoptosis by LPS.

Molecular cloning of the canine telomerase reverse transcriptase gene and its expression in neoplastic and non-neoplastic cells.

OBJECTIVE: To perform molecular cloning of the canine telomerase reverse transcriptase (TERT) gene and determine its expression in neoplastic and non-neoplastic cells. SAMPLE POPULATION: 9 canine tumor cell lines derived from various neoplasms, 16 primary canine tumors, and tissues from 15 normal canine organs. PROCEDURE: Tumor cell lines were derived from canine tumors that included osteosarcoma, mammary gland adenocarcinoma, melanoma, acute lymphoblastic leukemia, lymphoma, and mastocytoma and a canine primary fibroblast culture. Canine TERT complementary DNA (cDNA) was amplified by use of polymerase chain reaction (PCR) and sequenced. Expression of TERT mRNA was examined by reverse transcription (RT)-PCR assay. Telomerase activity was measured by use of the telomeric repeat amplification protocol assay. RESULTS: The canine TERT cDNA clone was 237 base pairs in length and contained a central region encoding the reverse transcriptase motif 2. Expression of TERT mRNA was detected in canine tumor cell lines that had telomerase activity but not in telomerase-negative canine primary fibroblasts. The TERT mRNA was detected in 13 of 16 canine tumor tissues and several normal tissues such as liver, ovary, lymph node, and thymus. A significant correlation between TERT expression level and telomerase activity was noted. CONCLUSIONS AND CLINICAL RELEVANCE: Expression of TERT mRNA was closely associated with telomerase activity in neoplastic cells as well as some non-neoplastic cells from dogs. In addition to telomerase activity, expression of TERT mRNA can be used as a marker of tumor cells.

Effect of an adenoviral vector that expresses the canine p53 gene on cell growth of canine osteosarcoma and mammary adenocarcinoma cell lines.

OBJECTIVE: To generate an adenoviral vector that expressed the canine p53 gene and investigate its growth-inhibiting effect on canine osteosarcoma and mammary adenocarcinoma cell lines. SAMPLE POPULATION: 2 canine osteosarcoma cell lines (HOS, OOS) and 3 canine mammary adenocarcinoma cell lines (CHMp, CIPm, and CNMm). PROCEDURE: An adenoviral vector that expressed the canine p53 gene (AxCA-cp53) was generated. p53 gene expression was examined by use of reverse transcription (RT)-polymerase chain reaction (PCR) assay and immunohistochemistry. Susceptibility of cell lines to the adenoviral vector was determined by infection with an adenoviral vector that expresses beta-galactosidase (AxCA-LacZ) and 3-indolyl-beta-D-galactopyranoside staining. Growth inhibitory effects were examined by monitoring the numbers of cells after infection with mock (PBS) solution, AxCA-LacZ, or AxCA-cp53. The DNA contents per cell were measured by flow cytometry analysis. Apoptotic DNA fragmentation was detected by use of a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. RESULTS: AxCA-cp53-derived p53 gene mRNA and P53 protein were detected by RT-PCR analysis and immunohistochemistry, respectively. Multiplicity of infection at which 50% of cells had positive 3-indolyl-beta-D-galactopyranoside staining results ranged from 10 to 50. AxCA-cp53 induced growth inhibition in a dose-dependent manner. Arrest of the G1-phase population and apoptotic DNA fragmentation were observed in cells infected with AxCA-cp53. CONCLUSIONS AND CLINICAL RELEVANCE: AxCA-cp53 inhibits cell growth via induction of cell cycle arrest and apoptosis in canine osteosarcoma and mammary adenocarcinoma cell lines that lack a functional p53 gene. AxCA-cp53 may be useful to target the p53 gene in the treatment of dogs with tumors.

Molecular cloning of the feline telomerase reverse transcriptase (TERT) gene and its expression in cell lines and normal tissues.

Telomerase is a kind of reverse transcriptase which synthesizes and elongates telomeres. Telomerase activity is detected in many naturally occurring tumors and its expression appears to play an important role in the immortalization of tumor cells. In this study, cDNA encoding the feline telomerase reverse transcriptase (TERT) gene was cloned partially from a feline lymphoma cell line. The clone obtained in this study was 237 bp long including a reverse transcriptase motif 2, and was shown to have amino acid sequence similarity of 81.0% and 58.2% with human and mouse TERT cDNAs, respectively. TERT mRNA expression was detected in telomerase-positive cells (FL74, FT-1, 3201, FKNp, FONp, and FYMp), and was not detected in telomerase-negative cells (normal fibroblasts and CRFK). TERT mRNA was detected in various normal tissues including the spleen, pancreas, stomach, cerebrum, testis, bone marrow, lymph node and thymus, and relatively high-level expression was observed in the small bowel and large bowel. No expression of TERT mRNA was detected in the liver, adrenal gland, urinary bladder and lung. The TERT cDNA clone and the results obtained in this study will be useful for further investigation of feline tumors.

Nucleotide sequence and expression of the feline vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is an angiogenic factor which targets vascular endothelial cells. In this study, cDNA encoding a feline VEGF (fVEGF) isoform was cloned from a feline lymphoid tumor cell line and sequenced. The fVEGF cDNA contained an open reading frame of 567 nucleotides coding for a polypeptide of 163 amino acids with a putative signal peptide of 26 amino acids. The predicted fVEGF amino acid sequence shared 98.4, 94.2 and 94.2% homology with the sequences of canine, bovine and human VEGF, respectively. Though predicted fVEGF polypeptide was two amino acid residues shorter than human VEGF165, a potential glycosylation site and regions critical for receptor binding were conserved in all the species examined. Transient expression of fVEGF in mammalian cells resulted in secretion of VEGF which could be detected by antibodies against human VEGF165. Furthermore, wide expression of fVEGF mRNA was observed in various feline tissues using RT-PCR methods.