Generation of anti-Notch antibodies and their application in blocking Notch signalling in neural stem cells.

Notch signalling occurs via direct cell-cell interactions and plays an important role in linking the fates of neighbouring cells. There are four different mammalian Notch receptors that can be activated by five cell surface ligands. The ability to inhibit specific Notch receptors would help identify the roles of individual family members and potentially provide a means to study and control cell differentiation. Anti-Notch antibodies in the form of single chain Fvs were generated from an antibody phage display library by selection on either the ligand binding domain or the negative regulatory region (NRR) of Notch1 and Notch2. Six antibodies targeting the NRR of Notch1 and four antibodies recognising the NRR of Notch2 were found to prevent receptor activation in cell-based luciferase reporter assays. These antibodies were potent, highly specific inhibitors of individual Notch receptors and interfered with endogenous signalling in stem cell systems of both human and mouse origin. Antibody-mediated inhibition of Notch efficiently down-regulated transcription of the immediate Notch target gene hairy and enhancer of split 5 (Hes5) in both mouse and human neural stem cells and revealed a redundant regulation of Hes5 in these cells as complete down-regulation was seen only after simultaneous blocking of Notch1 and Notch2. In addition, these antibodies promoted differentiation of neural stem cells towards a neuronal fate. In contrast to the widely used small molecule γ-secretase inhibitors, which block all 4 Notch receptors (and a multitude of other signalling pathways), antibodies allow blockade of individual Notch family members in a highly specific way. Specific inhibition will allow examination of the effect of individual Notch receptors in complex differentiation schemes regulated by the co-ordinated action of multiple signalling pathways.

Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse.

We describe the 'Crescendo Mouse', a human VH transgenic platform combining an engineered heavy chain locus with diverse human heavy chain V, D and J genes, a modified mouse Cγ1 gene and complete 3' regulatory region, in a triple knock-out (TKO) mouse background devoid of endogenous immunoglobulin expression. The addition of the engineered heavy chain locus to the TKO mouse restored B cell development, giving rise to functional B cells that responded to immunization with a diverse response that comprised entirely 'heavy chain only' antibodies. Heavy chain variable (VH) domain libraries were rapidly mined using phage display technology, yielding diverse high-affinity human VH that had undergone somatic hypermutation, lacked aggregation and showed enhanced expression in E. coli. The Crescendo Mouse produces human VH fragments, or Humabody® VH, with excellent bio-therapeutic potential, as exemplified here by the generation of antagonistic Humabody® VH specific for human IL17A and IL17RA.

Dendritic cells internalise and re-present conformationally intact soluble MHC class I alloantigen for generation of alloantibody.

Following organ transplantation soluble MHC class I is released from the graft and may contribute to alloimmunity. We determined in a well-established rat model whether DC are able to internalise soluble MHC class I alloantigen and then re-present intact alloantigen to B cells and T cells for generation of an alloantibody or CD8 T cell response. PVG.RT1(u) BM-derived DC internalised (via an active process) and retained intact a recombinant soluble form of RT1-A(a) (sRT1-A(a)). When PVG.RT1(u) rats were immunised with sRT1-A(a)-pulsed syngeneic DC, they developed a strong anti-sRT1-A(a) alloantibody response and showed accelerated rejection of RT1-A(a)-disparate PVG.R8 heart grafts. Alloantibody production and accelerated heart graft rejection were both dependent on immunisation with viable sRT1-A(a)-pulsed DC. The alloantibody response to sRT1-A(a)-pulsed DC was directed exclusively against conformational epitopes expressed by sRT1-A(a) and not epitopes expressed, for example, by non-conformational sRT1-A(a) heavy chain. Immunisation with sRT1-A(a)-pulsed syngeneic DC did not stimulate a CD8 T cell response. Our findings suggest a novel alloantigen recognition pathway whereby soluble MHC class I alloantigen released from an allograft may be taken up by recipient DC and presented in an intact unprocessed form to B cells for the generation of an alloantibody response.

Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.

Uptake and processing of Chlamydia trachomatis by human dendritic cells.

Chlamydia trachomatis (CT) causes several sexually transmitted diseases. In 2-5% of cases, CT infection leads to the development of reactive arthritis. Dendritic cells (DC) are central in T cell priming and the induction of antigen specific immunity. Here we have studied the uptake and processing of CT serovar L2 by human DC, and their ability to present CT antigens to both CD4(+) and CD8(+) T cells. We show that the entry of CT was mediated by the attachment of CT to heparan sulfates and could be inhibited by heparin. There was no inhibition of uptake by an agent which blocks micropinocytosis. Infecting DC with CT led to their activation and the production of IL-12 and TNF-alpha but not IL-10. Following invasion, CT was confined to distinct vacuoles which were visualized with anti-CT antibodies using confocal microscopy. Unlike with epithelial cells, these vacuoles did not develop into characteristic inclusion bodies. In the first 48 h, CT(+) vacuoles were negative for Lamp-1 and MHC class II. Despite no obvious co-localization between CT vacuoles and MHC loading compartments, infected DC efficiently presented CT antigens to CD4(+) T cells. Infected DC also expanded CT specific CD8(+) T cells, allowing us to generate a number of CT-reactive CD8(+) T cell clones. There is still controversy about the importance of chlamydia-specific CD8(+) T cell responses in patients with arthritis. This is largely due to the difficulties in studying CTL responses at the clonal level. The use of DC as antigen-presenting cells should enable more detailed characterization of these CTL responses.