immunocompetent recipient. The recombinant derived LDL receptor protein may be new to the 
recipient, and therefore self-tolerance to these proteins may not have developed. This opens the 
possibility that the recipient could make an immune response to these new proteins that could 
destroy the transplanted cells. In addition, changes in the transfected cells, such as 
inappropriate expression of Class II HLA antigens, could cause the recipients to develop an 
immune response to hepatocyte proteins in a manner similar to autoimmunity. 
Arguing against these latter two consequences are the results seen in autologous cell 
transfers in rabbits, where no immune response to the LDL receptor was noted. However, in 
other systems, such as the transfection of human growth hormone genes with autologous rat 
fibroblasts (113), immune responses to the transfected protein have occurred. Because of this 
example and the differences between the human and rabbit immune system, the possibility of an 
immune response to neoantigens on the transferred hepatocytes remains an important concern 
and immune responses to the LDL receptor, and the transduced hepatocytes will have to be 
monitored throughout the course of the cell transplant. 
' 2. Monitoring and Detection of Immune Responses to 
Transduced Ceil Proteins 
Immune responses to several different types of antigens will be monitored throughout 
the course of the protocol. 
Serologic Assays. Antibodies to the LDL receptor will be measured by two different 
techniques. Initial serologic reactivity to the LDL receptor will be assayed by Western blots of 
FH fibroblasts transfected with the LDL receptor and by immunoprecipitation in in vitro 
translated whole LDL receptor. Presence of glycosylated epitopes will be determined by 
evaluating Western blot reactivity to membranes treated with periodate. Localization of 
antibody epitopes in the LDL receptor will be performed by immunoprecipitation of carboxyl 
terminal truncations of the receptor produced by in vitro translation or by reactivity with 
fragments of the LDL receptor protein produced in a bacterial expression system. Due to the 
relative insensitivity of the Western blot assay, a dual capture ELISA assay for antibodies to 
LDL receptor also will be employed. This assay uses a mouse monoclonal antibody(1 1 5,1 1 6) to 
bind the LDL receptor onto the wells of an ELISA plate. After the non-specific proteins are 
washed off, human sera is added and specific antibodies will bind to the immobilized LDL 
receptor. The bound antibody will then by detected with an enzyme conjugated second antibody. 
This assay is more sensitive than the Western blot assays but shows slightly less specificity. In 
consideration of this, both LDL receptor-antibody assays will be performed. These studies will 
be done, pre-transplant and at one week intervals post transplant for approximately 90 days. 
Many of these serologic techniques were developed and have been employed to measure 
immune responses in gene transfer experiments in the WHHL rabbit model of FH. Because 
conformation and glycosylation determinants of the LDL receptor may be involved as epitopes in 
an antibody response, it was decided to develop assays that use whole LDL receptor preparations 
produced in eukaryotic cells. Fibroblasts from FH patients (lacking a native LDL receptor) 
were transfected with the rabbit LDL receptor and grown in culture. Membrane preparations 
made from these cells were then electrophoresed in SDS-PAGE under non-reducing conditions, 
and transferred to nitrocellulose. The transfers were then probed with sera from WHHL 
rabbits that had received a transplant of autologous hepatocytes transfected with rabbit LDL 
receptor. Control blots were performed in parallel using fibroblasts transfected with a control 
vector. Results of these studies suggested that rabbits having long-term decreases in serum 
lipid levels did not develop serologic responses to the receptor. Additional studies are being 
conducted to localize serologic determinants in the receptor. To do this, the receptor has been 
place in a Bluescript II vector for use in immunoprecipitation studies. This plasmid will be cut 
at various locations throughout the LDL receptor coding region, transcribed in vitro, and 
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Recombinant DNA Research, Volume 15 
