446 The severe shortage of allogeneic organs available for human transplantation has led to the consideration of the use of pigs as organ donors. Human preformed and induced antibody responses to pig xenografts, however, present a major barrier to the successful use of pig organs for human transplantation. We have recently identified the IgVH genes responsible for encoding IgM xenoantibody responses in humans exposed to pig tissues in a bioartificial liver support device. These responses are encoded by Ig genes that are members of the VH3 family and are restricted to genes encoded by the IgHV3-11 and IGHV3-74 germline progenitors. Induced xenoantibody responses in human patients at 10 days post BAL exposure demonstrate a clonal expansion of a VH gene with a unique VDJ gene configuration. We have recently demonstrated that this clonally expanded gene encodes a functional antibody that reacts with the α-gal epitope. In this report we have extended our analysis of the host response to the Ig genes used to encode IgG xenoantibodies. We have generated IgG gene libraries established from lymphocytes of patients at days 0 and 22 following treatment with a bioartificial liver containing pig hepatocytes and endothelial cells. cDNA libraries representing the VH3 family were screened by colony filter hybridization to identify increases in the frequency of expression of specific VH genes encoding α-gal antibodies. Immunoglobulin genes derived from the IGHV3-11 germline progenitor demonstrate an increase in frequency of expression from 2.9% at day 0 to 20% at day 22. A CDR3 specific probe was used to identify an IgM to IgG isotype switch during the maturation of the antibody response. A unique, clonally expanded IGHV3-11 gene identified in IgM clones at day 10 represent the majority of IgG clones expressed at high levels at day 22. The identify of this clone was confirmed by DNA sequencing. IgVH genes encoded by the IGHV3-74 family demonstrate an increase in expression from 0.8% at day 0 to 5.5% at day 22. Genes related to V3-7 germline progenitors do not contribute to the xenoantibody response. These results demonstrate that the xenoantibody response in humans is encoded by IgVH genes restricted to IGHV3-11 and IGHV3-74 germline families. A specific xenoantibody reactive with the α-gal epitope undergoes a clonal expansion and an isotype switch during the maturation of the xenoantibody response. The restricted nature of the humoral response to pig tissues is of potential importance for the development of therapeutic strategies to prevent xenograft rejection.

Preformed and induced Ab responses present a major immunological barrier to the use of pig organs for human xenotransplantation. We generated IgM and IgG gene libraries established from lymphocytes of patients treated with a bioartificial liver (BAL) containing pig hepatocytes and used these libraries to identify IgVH genes that encode human Ab responses to pig xenoantigens. Genes encoded by the VH3 family are increased in expression in patients following BAL treatment. cDNA libraries representing the VH3 gene family were generated, and the relative frequency of expression of genes used to encode the Ab response was determined at days 0, 10, and 21. Ig genes derived from the IGHV3-11 and IGHV3-74 germline progenitors increase in frequency post-BAL. The IGHV3-11 gene encodes 12% of VH3 cDNA clones expressed as IgM Abs at day 0 and 32.4-39.0% of cDNA clones encoding IgM Abs in two patients at day 10. IGHV3-11 and IGHV3-74 genes encoding IgM Abs in these patients are expressed without evidence of somatic mutation. By day 21, an isotype switch occurs and IGHV3-11 IgVH progenitors encode IgG Abs that demonstrate somatic mutation. We cloned these genes into a phagemid vector, expressed these clones as single-chain Abs, and demonstrated that the IGHV3-11 gene encodes Abs with the ability to bind to the gal alpha (1,3) gal epitope. Our results demonstrate that the xenoantibody response in humans is encoded by IgVH genes restricted to IGHV3-11 and IGHV3-74 germline progenitors. IgM Abs are expressed in germline configuration and IgG Abs demonstrate somatic mutations by day 21.

82 The use of pigs as organ donors for human transplantation has the potential to alleviate the severe shortage of suitable donor organs. Human preformed and induced antibody responses to pig xenografts present a major barrier to the successful use of xenografted organs for transplantation. In order to characterize the human immunoglobulin response to pig xenografts, we have generated IgM gene libraries established from lymphocytes of patients treated with a bioartificial liver containing pig hepatocytes and endothelial cells and used these libraries to identify IgVH genes used to encode human antibody responses to pig xenoantigens. An anchored PCR-ELISA assay was used to demonstrate the rapid expansion of VH3 family encoded immunoglobulin VH genes and a smaller increase in genes related to the VH4 family in two patients following BAL treatment. cDNA libraries representing the VH3 gene family were generated and screened by colony filter hybridization to identify increases in the frequency of expression of specific VH genes following exposure to pig cells. We have sequenced 68 genes from three different patients at days 0,1,10, and 21 following BAL treatment and have used the colony hybridization technique to examine the frequency of VH3 family genes known to encode antibodies that react with the α(1,3) gal epitope. Immunoglobulin genes related to the V3-7 and V3-74 germline progenitors increase in frequency in patients exposed to pig cells. VH genes encoded by the DP58 germline progenitor do not increase in frequency in these patients, suggesting that the response is specific and restricted to selected genes within the VH3 family. We have identified an additional VH3 gene that represents 12% of the VH3 gene expression at day 0 but rises to 32.4-39% in two patients following exposure to pig cells. Nucleic acid sequencing and colony filter hybridization using a CDR3 specific oligonucleotide probe has provided evidence for the clonal expansion of a specific VDJ gene configuration in the majority of clones at day 10 following pig xenoantigen exposure. Preliminary analysis of the structure of xenoantibody variable regions of the genes involved in patient xenoantibody responses and antibodies reactive with theα1,3 gal epitope suggests that these antibodies share identical amino acids that characterize the canonical structure of their hypervariable regions essential for determining antigen/antibody interaction. Our data suggests that human IgM antibodies to pig xenoantigens are a closely-related, structurally similar group of immunoglobulin genes including IgVH genes expressed in a germline configuration.