An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma.

Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 105 copies/ng DNA, and 88% of HPV mRNA expression was found to be E4, E5a, and E5b mRNAs. According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.

Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma.

Laryngeal papilloma (LP) associated with human papillomavirus (HPV)-6 or -11 infection shows aggressive growth. However, the detailed molecular mechanism of virus-driven tumorigenesis has not been uncovered fully. HPV-6 viral gene expression and dynamic alterations were investigated with in situ localization of viral DNA and RNA in 13 patients with HPV-6-infected laryngeal papilloma. The average viral load was 4.80 × 105 ± 1.86 × 105 copies/ng DNA. E4, E5a, and E5b mRNAs accounted for 96% of the expression of 9 mRNAs. The alteration of viral DNA load during recurrence paralleled the mRNA expression levels, and the expression of all mRNAs showed a similar curve. E4, E5a, and E5b were expressed in the middle to upper part of the epithelium and were co-expressed in the same cells. E4 immunohistochemistry demonstrated an extensively positive reaction in the upper cell layer in accordance with E4 mRNA expression. These results suggest that individual viral genes are coordinately expressed for viral replication, virus release, and immunosurveillance avoidance. The newly developed E4-specific monoclonal antibody can be applied to further functional studies and clinical applications such as targeted molecular therapies.

Adipose tissue-derived mesenchymal stem cells ameliorate bone marrow aplasia related with graft-versus-host disease in experimental murine models.

Graft-versus-host disease (GVHD) constitutes the most frequent complications after the allogeneic hematopoietic stem cell transplantation for a variety of hematological malignancies. In the present study, we explored the prophylactic potential of adipose tissue-derived mesenchymal stem cells (AD-MSCs) in controlling GVHD in murine models with a special focus on bone marrow aplasia related with acute GVHD. The CB6F1 mice were induced GVHD by the injection intravenously of C57BL/6 (B6-Ly-5.1) splenocytes without conditioning irradiation or chemotherapy. AD-MSCs from C3H mice were injected intravenously via tail veins. GVHD was assessed using flowcytometry analysis of peripheral blood cells and histopathologic analysis of target organs. Histopathological analyses revealed that AD-MSCs markedly suppressed the infiltration of lymphocytes into liver as well as the aplasia in bone marrow. This study is the first to clarify the effectiveness of AD-MSCs against bone marrow aplasia in GVHD, supporting a rationale of AD-MSCs for ameliorating bone marrow suppression and infectivity after allo-HSCT in human clinics.

Lymphopenia induced by a novel selective S1P(1) antagonist structurally unrelated to S1P.

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P(1)) and participates in many pathological conditions. We developed a novel type S1P(1)-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P(1) resulting in reduced signaling downstream of S1P(1), including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P(1)-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P(1) antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P(1) agonists on lymphocyte sequestration results from their functional antagonism.

Amelioration of collagen-induced arthritis by a novel S1P1 antagonist with immunomodulatory activities.

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking through the type 1 sphingosine 1-phosphate receptor (S1P(1)) and participates in many pathological conditions, including autoimmune diseases. We developed a novel S1P(1)-selective antagonist, TASP0277308, which is structurally unrelated to S1P. This antagonist competitively inhibited S1P-induced cellular responses, such as chemotaxis and receptor internalization. Furthermore, differing from previously reported S1P(1) antagonists, TASP0277308 demonstrated in vivo activities to induce lymphopenia, a block in T cell egress from the thymus, displacement of marginal zone B cells, and upregulation of CD69 expression on both T and B cells, all of which recapitulate phenotypes of S1P(1)-deficient lymphocytes. In a mouse collagen-induced arthritis model, TASP0277308 significantly suppressed the development of arthritis, even after the onset of disease. These findings provide the first chemical evidence to our knowledge that S1P(1) antagonism is responsible for immunosuppression in the treatment of autoimmune diseases and also resolve the discrepancies between genetic and chemical studies on the functions of S1P(1) in lymphocytes.