Selective Inhibition of ADAM28 Suppresses Lung Carcinoma Cell Growth and Metastasis.

ADAM28 (a disintegrin and metalloproteinase 28) is overexpressed by carcinoma cells in non-small cell lung carcinomas (NSCLC) and plays an important role in cancer cell proliferation and metastasis by reactivation of insulin-like growth factor-1 (IGF-1) and escaping from von Willebrand factor (VWF)-induced apoptosis through digestion of IGF-binding protein-3 and VWF, respectively. To aim for new target therapy of NSCLC patients, we developed human neutralizing antibodies 211-12 and 211-14 against ADAM28, which showed IC50 values of 62.4 and 37.5 nmol/L, respectively. Antibody 211-14 recognized the junctional region between cysteine-rich domain and secreted-specific domain and showed a KD value of 94.7 pmol/L for the epitope-containing peptide. This antibody detected monkey and human secreted-form ADAM28s, although it was not reactive with mouse membrane-anchored ADAM28m. Antibody 211-14 effectively inhibited IGF-1-stimulated cell proliferation of lung adenocarcinoma cell lines with ADAM28 expression, including PC-9 cells, and promoted VWF-induced cell death in these cell lines. In lung metastasis models, antibody 211-14 significantly reduced tumor growth and metastases of PC-9 cells and prolonged survivals in the antibody-treated mice compared with the control IgG-treated ones. Combination therapy of the antibody and docetaxel was more effective than that of bevacizumab and docetaxel and showed further elongation of survival time compared with monotherapy. No adverse effects were observed even after administration of 10-fold more than effective dose of anti-ADAM28 antibody to normal mice. Our data demonstrate that antibody 211-14 is a neutralizing antibody specific to ADAM28s and suggest that this antibody may be a useful treatment remedy for NSCLC patients. Mol Cancer Ther; 17(11); 2427-38. ©2018 AACR.

Development of human neutralizing antibody to ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2).

ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2), members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, are considered to play a key role in aggrecan degradation of articular cartilage in human osteoarthritis. Here, we developed a neutralizing antibody to these aggrecanases by screening human combinatorial antibody library. Among the five candidate antibodies, one antibody was immunoreactive with both ADAMTS4 and ADAMTS5, showing no or negligible cross-reactivity with 10 different related metalloproteinases of the ADAMTS, ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) gene families. This antibody almost completely and partially inhibited aggrecanase activity of ADAMTS4 and ADAMTS5, respectively. It also suppressed the aggrecanase activity derived from interleukin-1-stimulated osteoarthritic chondrocytes. These data demonstrate that the antibody is specific to ADAMTS4 and ADAMTS5 and inhibits their aggrecanase activity at molecular and cellular levels, and suggest that this antibody may be useful for treatment of pathological conditions such as osteoarthritis.

Nonviral Abeta DNA vaccine therapy against Alzheimer's disease: long-term effects and safety.

It was recently demonstrated that amyloid beta (Abeta) peptide vaccination was effective in reducing the Abeta burden in Alzheimer model mice. However, the clinical trial was halted because of the development of meningoencephalitis in some patients. To overcome this problem, anti-Abeta antibody therapy and other types of vaccination are now in trial. In this study, we have developed safe and effective nonviral Abeta DNA vaccines against Alzheimer's disease. We administered these vaccines to model (APP23) mice and evaluated Abeta burden reduction. Prophylactic treatments started before Abeta deposition reduced Abeta burden to 15.5% and 38.5% of that found in untreated mice at 7 and 18 months of age, respectively. Therapeutic treatment started after Abeta deposition reduced Abeta burden to approximately 50% at the age of 18 months. Importantly, this therapy induced neither neuroinflammation nor T cell responses to Abeta peptide in both APP23 and wild-type B6 mice, even after long-term vaccination. Although it is reported that other anti-Abeta therapies have pharmacological and/or technical difficulties, nonviral DNA vaccines are highly secure and easily controllable and are promising for the treatment of Alzheimer's disease.

Characterization of relapsing autoimmune encephalomyelitis and its treatment with decoy chemokine receptor genes.

To elucidate the pathomechanisms of relapses of autoimmune disorders and to develop immunotherapy against relapses, we induced acute monophasic and chronic relapsing (CR) experimental autoimmune encephalomyelitis (EAE) in DA rats. Immunopathological and cytokine-chemokine analyses demonstrated that the number of infiltrating macrophages was significantly elevated in the CR-EAE than in acute EAE lesions and that IFN-gamma and IP-10 in the spinal cord were significantly upregulated during the first attack and relapse of CR-EAE, respectively, than at the peak of acute EAE. In vivo administration of decoy chemokine receptor plasmid DNAs encoding the binding sites of CXCR3 and CCR2 suppressed the development of relapse of CR-EAE. Importantly, multiple injections of DNAs did not elicit the antibody production against chemokine receptors. Taken together, these findings demonstrated that neutralization therapy with decoy chemokine receptor DNAs is effective to control autoimmune diseases.

C protein-induced myocarditis and subsequent dilated cardiomyopathy: rescue from death and prevention of dilated cardiomyopathy by chemokine receptor DNA therapy.

Severe experimental autoimmune myocarditis and subsequent dilated cardiomyopathy (DCM) were successfully produced in Lewis rats by immunization with recombinant cardiac C protein. Seventy-five percent of immunized rats died between days 15 and 49 postimmunization, and all of the survived rats showed typical DCM characterized by the presence of ventricular dilatation and extensive fibrosis. Immunopathological and chemokine analysis during the acute phase revealed that there were marked macrophage infiltration with myocyte necrosis and up-regulation of MCP-1 and IFN-gamma-inducible protein-10 (IP-10). Based on these findings, we prepared plasmid DNAs encoding the binding site of CCR2 and CXCR3, which are receptors for MCP-1 and IP-10, respectively. The culture supernatant of cells transfected with these DNAs inhibited the migration of T cells and macrophages induced by MCP-1 and IP-10. Remarkably, administration of the DNAs to C protein-immunized rats prevented the disease progression and rescued animals from death. The present study has demonstrated for the first time that gene therapy targeting the chemokine receptor could be a powerful tool for the control of experimental autoimmune myocarditis and DCM.

Clinicopathological study of a myelin oligodendrocyte glycoprotein-induced demyelinating disease in LEW.1AV1 rats.

Although multiple sclerosis is considered to be an autoimmune disease in the CNS, the immune responses that take place in the CNS and lymphoid organs remain to be elucidated. Here, we have successfully induced various subtypes of experimental autoimmune encephalitis (EAE) in LEW.1AV1 rats carrying RT1(av1) on the Lewis background genes by immunization with recombinant rat myelin oligodendrocyte glycoprotein (MOG) in various solutions with adjuvants. The purpose of the present study was to analyse in more detail the clinical and immunopathological features of MOG-induced EAE in LEW.1AV1 rats. Immunization with high doses of soluble MOG with pertussis toxin induced acute, frequently fatal EAE, whereas medium doses of partially aggregated MOG without pertussis toxin produced relapsing and remitting EAE. Secondary progressive EAE was induced in some rats by immunization with the immunization protocol having an intermediate nature between the above two. The optic nerve (approximately 60% of the immunized rats) and spinal cord (100%) were frequently involved and detectable both clinically and pathologically, while there was no lesion in the cerebrum. Histological examination revealed that, despite variety in the clinical subtypes, progression of the pathological processes was strikingly uniform, i.e. initial inflammation with minimal demyelination followed by predominant demyelination with minimal lymphocyte infiltration. These findings suggest that the lesion during the later stage is maintained by humoral factors. Taken together, this experimental system can serve as a model of neuromyelitis optica. Further analysis will provide useful information to elucidate the pathogenesis and to develop immunotherapy for neuromyelitis optica and multiple sclerosis.

Rho guanine nucleotide exchange factor xNET1 implicated in gastrulation movements during Xenopus development.

During Xenopus development, embryonic cells dramatically change their shape and position. Rho family small GTPases, such as RhoA, Rac, and Cdc42, play important roles in this process. These GTPases are generally activated by guanine nucleotide exchange factors (GEFs); however, the roles of RhoGEFs in Xenopus development have not yet been elucidated. We therefore searched for RhoGEF genes in our Xenopus EST database, and we identified several genes expressed during embryogenesis. Among them, we focused on one gene, designated xNET1. It is similar to mammalian NET1, a RhoA-specific GEF. An in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. In addition, transient expression of xNET1 activated endogenous RhoA. These results indicated that xNET1 is a GEF for RhoA. Epitope-tagged xNET1 was localized mainly to the nucleus, and the localization was regulated by nuclear localization signals in the N-terminal region of xNET1. Overexpression of either wild-type or a mutant form of xNET1 severely inhibited gastrulation movements. We demonstrated that xNET1 was co-immunoprecipitated with the Dishevelled protein, which is an essential signaling component in the non-canonical Wnt pathway. This pathway has been shown to activate RhoA and regulate gastrulation movements. We propose that xNET1 or a similar RhoGEF may mediate Dishevelled signaling to RhoA in the Wnt pathway.

PKC delta is essential for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements.

Protein kinase C (PKC) has been implicated in the Wnt signaling pathway; however, its molecular role is poorly understood. We identified novel genes encoding delta-type PKC in the Xenopus EST databases. Loss of PKC delta function revealed that it was essential for convergent extension during gastrulation. We then examined the relationship between PKC delta and the Wnt pathway. PKC delta was translocated to the plasma membrane in response to Frizzled signaling. In addition, loss of PKC delta function inhibited the translocation of Dishevelled and the activation of c-Jun N-terminal kinase (JNK) by Frizzled. Furthermore, PKC delta formed a complex with Dishevelled, and the activation of PKC delta by phorbol ester was sufficient for Dishevelled translocation and JNK activation. Thus, PKC delta plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled.

Characterization of the antigen specificity and TCR repertoire, and TCR-based DNA vaccine therapy in myelin basic protein-induced autoimmune encephalomyelitis in DA rats.

Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62-75 sequence (MBP(62-75)) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP(62-75) was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that Vbeta10 and Vbeta15 spectratype expansion was always found in MBP(62-75)-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of Vbeta10- and Vbeta15-positive T cells. First, the adoptive transfer experiments revealed that Vbeta10-positive T line cells derived from MBP(62-75)-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding Vbeta10 and Vbeta15, alone or in combination, ameliorated MBP(62-75)-induced EAE. Collectively, it was strongly suggested that Vbeta10- and Vbeta15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases.