BACKGROUND: Respiratory syncytial virus (RSV) infection is a major problem in the newborn and aging populations. Fully human monoclonal antibodies with the ability to neutralize RSV could have a major impact on the immunotherapy of the disease. The generation of human antibodies has been difficult because there exists no general way to activate B cells against an antigen of choice in vitro. MATERIALS AND METHODS: Human spleen cells from individuals exposed to RSV were used to repopulate SCID mice. Hu-SCID mice were boosted with RSV fusion (F)-protein and subsequently developed B cell tumors. The tumors were removed and cultured and subcloned in vitro, using a feeder layer of CD154-expressing 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 was necessary to use high-temperature reverse transcriptase to clone the VH genes. RESULTS: RF-1 and RF-2 VH genes were both found to be closely related members of the VH2 family. Vk genes originated from the VK 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, respectively, 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, provides a method by which high-affinity human antibodies can be developed for immunotherapy of viral diseases.
BACKGROUND:Respiratory syncytial virus (RSV) infection is a major problem in the newborn and aging populations. Fully human monoclonal antibodies with the ability to neutralize RSV could have a major impact on the immunotherapy of the disease. The generation of human antibodies has been difficult because there exists no general way to activate B cells against an antigen of choice in vitro. MATERIALS AND METHODS:Human spleen cells from individuals exposed to RSV were used to repopulate SCIDmice. Hu-SCIDmice were boosted with RSV fusion (F)-protein and subsequently developed B cell tumors. The tumors were removed and cultured and subcloned in vitro, using a feeder layer of CD154-expressing 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 was necessary to use high-temperature reverse transcriptase to clone the VH genes. RESULTS:RF-1 and RF-2 VH genes were both found to be closely related members of the VH2 family. Vk genes originated from the VK 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, respectively, and both were able to neutralize several A and B subtypes of RSV. CONCLUSION: The technique of immortalizing human B lymphocytes, by passage in SCIDmice and expression as recombinant antibodies in CHO cells, provides a method by which high-affinity human antibodies can be developed for immunotherapy of viral diseases.
Authors: M D Sheets; P Amersdorfer; R Finnern; P Sargent; E Lindquist; R Schier; G Hemingsen; C Wong; J C Gerhart; J D Marks; E Lindqvist Journal: Proc Natl Acad Sci U S A Date: 1998-05-26 Impact factor: 11.205
Authors: C Queen; W P Schneider; H E Selick; P W Payne; N F Landolfi; J F Duncan; N M Avdalovic; M Levitt; R P Junghans; T A Waldmann Journal: Proc Natl Acad Sci U S A Date: 1989-12 Impact factor: 11.205
Authors: C F Barbas; J E Crowe; D Cababa; T M Jones; S L Zebedee; B R Murphy; R M Chanock; D R Burton Journal: Proc Natl Acad Sci U S A Date: 1992-11-01 Impact factor: 11.205
Authors: Larry Zeitlin; Ognian Bohorov; Natasha Bohorova; Andrew Hiatt; Do H Kim; Michael H Pauly; Jesus Velasco; Kevin J Whaley; Dale L Barnard; John T Bates; James E Crowe; Pedro A Piedra; Brian E Gilbert Journal: MAbs Date: 2013-02-08 Impact factor: 5.857