Two neutralizing human anti-RSV antibodies: Cloning, expression, and characterization

Background: Respiratory syncytial virus (RSV) infection is a major problem in the newborn and aging populations. Fully human monoclonal antibodies wit h the ability to neutralize RSV could have a major impact on the immunother apy of the disease. The generation of human antibodies has been difficult b ecause there exists no general way to activate B cells against an antigen o f choice in vitro. Materials and Methods: Human spleen cells from individuals exposed to RSV w ere used to repopulate SCID mice. Hu-SCID mice were boosted with RSV fusion (F)protein and subsequently developed B cell tumors. The tumors were remov ed and cultured and subcloned in vitro, using a feeder layer of CD154-expre ssing T cells. Two of these tumors produced the antibodies designated RF-1 and RF-2. VL genes were isolated by standard PCR techniques, however, it wa s necessary to use high-temperature reverse transcriptase to clone the VH g enes. Results: RF-1 and RF-2 VH genes were both found to be closely related membe rs of the VH2 family. Vk genes originated from the VI( III family. RF-1 and RF-2 recombinant antibodies expressed in CHO cells (cRF-1 and cRF-2) were found to have affinities for RSV F-protein of 0.1 nM and 0.07 nM, respectiv ely, and both were able to neutralize several A and B subtypes of RSV. Conclusion: The technique of immortalizing human B lymphocytes, by passage in sCID mice and expression as recombinant antibodies in CHO cells, provide s a method by which high-affinity human antibodies can be developed for imm unotherapy of viral diseases.