Anti-RSV Recombinant Antibody (Palivizumab) (CAT#: TAB-009)

Figure 1 RSV neutralization, F glycoprotein recognition, and crystal structure of human antibody D25 in complex with the prefusion RSV F trimer.

The prefusion conformation of RSV F is metastable, and when expressed in a soluble form readily adopts the postfusion state; a number of potent antibodies, including D25, bind to a newly revealed antigenic site at the top of the prefusion F glycoprotein. (A) RSV neutralization by antibodies. Palivizumab is the U.S. Food and Drug Administration (FDA)–approved prophylactic antibody thatprevents severe RSV disease. (B) Enzyme-linked immunosorbent assay measuring antibody binding to postfusion F glycoprotein. For (A) and (B), data are representative of multiple independent experiments. (C)D25-RSVFtrimercrystal structure in ribbon and molecular surface representations. One protomer of the F glycoprotein trimer is shown as ribbons and colored as a rainbow from blue to red, N terminus of F2to C terminus of F1, respectively. Molecular surfaces are shown for the other two F protomers, colored pink and green. The D25 Fab bound to the F protomer shown in ribbons is also displayed in ribbon representation, with the heavy chain colored red and light chain colored gray. The other D25 Fabs are colored the same, but shown in surface representation.

McLellan, J. S., Chen, M., Leung, S., Graepel, K. W., Du, X., Yang, Y., ... & Kumar, A. (2013). Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science, 340(6136), 1113-1117.

Figure 2 HighlyeffectiveRSVneutralizing antibodies target a site at the membranedistal apex of the prefusion F trimer.

(A) The ability of antibodies to block D25 binding to RSV-infected cells was measured as a function ofantibody concentration. (B)AnalysisofRSVF-Fabcomplexesbynegative-stain electron microscopy (EM): Reprojection of a 12 Å slice through the crystal structure of RSV F + D25 Fab filtered to 10 Å resolution (left). A slice was used to emphasize visibility of the F glycoprotein cavity. Aligned average of 263 particles of RSV F + D25 Fab (middle left). Aligned average of 550 particles of RSV F + AM22 Fab (middle right). Aligned average of 171 particles of RSV F + 5C4 Fab (right). Scale bar, 50 Å. (C) Fusion inhibition and (D)attachmentinhibition activity for antibodies targeting antigenic site Ø and F-specific antibodies targeting other antigenic sites. For the attachment-inhibition assay, heparin was usedasapositivecontrol.Datain(A),(C),and(D)are representative of multiple independent experiments.

McLellan, J. S., Chen, M., Leung, S., Graepel, K. W., Du, X., Yang, Y., ... & Kumar, A. (2013). Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science, 340(6136), 1113-1117.

Figure 3 Anti-RSV F antibodies identified from Morphosys HuCAL GOLD® phage display libraries.

Heavy chain C-terminal 6xHis tagged antigen specific bivalent Fabs were purified with Ni-NTA column and then tested in ELISA binding to RSV prefusion and postfusion F proteins. (A) Antibodies preferentially binding to RSV postfusion F protein; (B) Antibodies binding to both RSV postfusion and prefusion F proteins; (C) antibody binding specifically to RSV prefusion F protein. Full-length human IgG1 D25 (prefusion F specific) and palivizumab (binding to both prefusion and postfusion F) were used as control antibodies in the above experiments.

Chen, Z., Zhang, L., Tang, A., Callahan, C., Pristatsky, P., Swoyer, R., ... & DiStefano, D. (2016). Discovery and characterization of phage display-derived human monoclonal antibodies against RSV F glycoprotein. PloS one, 11(6), e0156798.

Figure 4 Characterization of mAbs 2E1 and 3B1.

(A-B) ELISA analysis of 2E1 IgG (A) and 3B1 IgG (B) binding to RSV pre- (red circle) and postfusion F (blue square) proteins. (C-H) Surface plasmon resonance (SPR) analysis of 2E1 and 3B1 Fabs binding to pre- and postfusion RSV F proteins. RU = Resonance Units. Monovalent Fab antibody fragments were captured on the surface of a Series S Sensor Chip CM5 previously functionalized with Human Fab Binder. Prefusion or postfusion F protein diluted 2-fold serially starting at 100 nM or 200 nM was then injected over captured 2E1 (C) or 3B1 (F), respectively. To determine steady-state affinity, response levels at equilibrium were plotted over concentration of pre- (D) or postfusion F (G) protein. 50 nM of post- (E) or prefusion F (H) was injected to demonstrate the specificity of 2E1 binding to prefusion F and 3B1 to postfusion F. (I-K) SPR based competition analysis of 2E1 and 3B1 against palivizumab (I), D25 (J) and MPE8 (K) in binding to RSV prefusion F protein. RU = Resonance Units. Palivizumab and D25 were amine coupled to the surface of separate flow channels of a CM5 chip. A third flow channel was subjected to amine coupling activation without an antibody and used for reference subtraction. Prefusion F (40 μg/mL) was then injected over all surfaces. After a brief stabilization period, 2E1, 3B1, or running buffer was injected to measure binding to sites not occupied by the capturing antibody (palivizumab or D25). To assess competition to MPE8, the MPE8 antibody was captured (6000 RU, not shown) to flow channel 2 of a Biacore Sensor Chip Protein A. Prefusion F (40 μg/mL) was passed over channels 1 and 2 followed by 2E1 Fab, 3B1 Fab, D25 Fab and buffer to measure binding to sites not occupied by MPE8. (L) Bio-Layer Interferometry (BLI) based competition experiment of 3B1 IgG against site I antibody 131-2a. Palivizumab (blue) is able to bind to postfusion F protein in an Octet sandwich competition assay using 131-2a as the capture antibody, but antibody 3B1 (red) does not bind.

Chen, Z., Zhang, L., Tang, A., Callahan, C., Pristatsky, P., Swoyer, R., ... & DiStefano, D. (2016). Discovery and characterization of phage display-derived human monoclonal antibodies against RSV F glycoprotein. PloS one, 11(6), e0156798.

Figure 5 Inactivation of RSV by UV or pretreatment with palivizumab (an anti-RSV F protein Ab) completely abolishes the expression of COX-2 mRNA and protein.

A, Analysis of COX-2 mRNA expression. For the UV-inactivated RSV study, cells were harvested at 24–48 h. Palivizumab-treated RSV (400 ug/ml palivizumab, 1 h incubation at room temperature) or control, untreated, RSV was incubated with the cells for 48 h. RSV F protein mRNA expression is shown as a control for viral replication. B, Western blot analysis of the expression of COX-2 protein. The virus preparations used were RSV alone (RSV), RSV inactivated by UV (UV-RSV), RSV incubated for 1 h with palivizumab (400 ug/ml), or RSV treated with a control unrelated mAb (C mAb RSV). RSV was used at an equivalent of 0.5 m.o.i. in all cases. Data shown are representative of four independent experiments.

Richardson, J. Y., Ottolini, M. G., Pletneva, L., Boukhvalova, M., Zhang, S., Vogel, S. N., ... & Blanco, J. C. (2005). Respiratory syncytial virus (RSV) infection induces cyclooxygenase 2: a potential target for RSV therapy. The Journal of Immunology, 174(7), 4356-4364.