Human monoclonal antibody technology. A tool to investigate human antibody repertoires
Sammanfattning: The development of technologies to generate cell lines producing monoclonal antibodies of human origin has a great impact on the ability to study repertoires of specificities in the humoral immune response. Furthermore, it permits the generation of reagents which may be valuable for diagnostic and therapeutic applications and which lack some of the disadvantages of similar murine antibodies. In this study Epstein-Barr virus (EBV)-transformation of peripheral blood mononuclear cells (PBMC) has been used to immortalize human B lymphocytes. A novel technique to eliminate the capacity of PBMC to inhibit the outgrowth of EBV-transformed B cells is described. This technique, which employs pretreatment of PBMC with L-leucyl-L-leucine methyl ester, removes the inhibitory effect mediated by T cell populations in vitro. Furthermore, it is shown that the treatment impairs the ability of CD4+ T lymphocytes to respond to EBV-infected B cells by production of mRNA encoding the outgrowth-inhibitory lymphokine interferon-gamma. This technique has been used to allow establishment of lymphoblastoid cell lines (LCL) producing antigen-specific human antibodies from PBMC obtained from non-immunized donors, following in vitro stimulation. The LCL were subsequently fused to an immortal heteromyeloma fusion partner in order to rescue the antibody specificities of the potentially unstable EBV-transformed cell lines. Monoclonal antibodies recognizing digoxin or recombinant fragments of HIV-1 related glycoproteins gp41/gp120 have thus been obtained and characterized with respect to fine specificity using epitope mapping. These antibodies are of the low-affinity IgM type although the genes encoding the immunoglobulin variable regions show evidence of mutations in relation to previously described germ line sequences. Despite the low affinity of these immunoglobulins, some of the HIV-1 gp120-specific antibodies are able to block spreading of virus between cells in vitro thus suggesting an ability of antibodies established by these approaches from naive/primary repertoires to mediate biologically important functions. Human monoclonal antibody-producing cell lines secreting immunoglobulins recognizing either phosphoprotein (pp65) or glycoprotein (gB) of cytomegalovirus (CMV) have similarly been established from lymphocytes derived from seropositive donors. The antigens recognized by these antibodies have previously been suggested to be important for the diagnosis of CMV infection and for the humoral immune surveillance of CMV-related infections, respectively. Conformations as well as apparently linear epitopes which are detected by human antibody repertoires have been characterized. In addition, the neutralizing capacity of antibodies recognizing different fine-specificities expressed by glycoprotein B is described.
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