Sequences of variable regions of a monoclonal antibody specific to the thyroid hormone, triiodo-L-thyronine.

We have determined the nucleotide sequences of the variable regions of H and L chains of a monoclonal antibody 98QQ that interacts with the thyroid hormone triiodo-L- thyronine with high affinity. Analysis of the nucleotide sequence of the light chain V region of 98QQ revealed that the VL sequence is 99% identical to Balb/c germline Vk 21-E sequence. That is an interesting finding with this high affinity anti-T3 antibody, since occurence of predominantly germline variable region sequences is observed in some autoantibodies to self antigens but not usually in high affinity IgG antibodies. The sequence analysis also revealed that the heavy chain variable region sequence of 98QQ is similar to a V region of an anti-DNA antibody (MRL DNA 22). Thus the sequence analysis of our anti-T3 mAb 98QQ has revealed some features of autoantibodies to self antigens.

Construction, expression and characterization of a murine/human chimeric antibody with specificity for hepatitis B surface antigen.

A murine/human chimeric antibody with specificity for Hepatitis B surface antigen has been produced by genetic engineering. The light and heavy chain variable region exons encoding the murine monoclonal antibody 2H1 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 constant region exons. The chimeric genes were transfected into murine Sp2/0 hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1-7 pg/cell/24 hr. The chimeric antibody bound specifically to Hepatitis B surface antigen and competed effectively with the parental murine monoclonal antibody for binding to these sites. Chimeric 2H1 is the first clinically relevant, genetically engineered anti-viral antibody and may represent an improved agent for the prevention of hepatitis B virus transmission.

An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells.

We employed reverse genetics to clone a 5.0 kb genomic DNA hot spot HIRPE (hot spot for increased recombinant protein expression) flanking the plasmid integration site from a recombinant Chinese hamster ovary (CHO) cell line. DNA sequence analysis of the 5.0 kb fragment revealed that HIRPE is enriched for repetitive elements, Alu-like sequences and matrix-associated regions that are known to be linked with transcriptionally active regions in a number of mammalian systems. The construction of a homologous recombination vector, pTV1, containing the 5.0 kb HIRPE genomic DNA, a recombinant gene human CTLA4-Ig, and the dhfr gene as a positive selection marker is described. It was observed that the pTV1 vector targeted the CTLA4Ig gene to a preferred locus in the CHO genome contributing to high recombinant gene expression in transfected CHO cells. Preliminary studies suggest that similar to the observation with the parental cell line, pTV1-generated transfectomas that were analyzed appear to harbor an inverted duplication of the genomic DNA at the plasmid integration site.

Phosphorylated proteins in Drosophila membranes.

Reversible protein modification-demodification in bacterial membranes has been shown to be an important mechanism for the adaptive behavior of bacteria in response to chemosensory stimuli. It has been suggested that the protein modification mechanisms might have wider functional implications and might form the basis for an understanding of complex phenomena such as information storage and retrieval. Phosphorylation of membrane proteins in the mammalian system is a well-documented phenomenon. Greengard and coworkers have shown that phosphorylation of a set of synaptic membrane proteins, collectively known as protein I is stimulated specifically in response to cAMP and calcium. We have explored the possibility of in vitro phosphorylation of proteins in membrane preparations obtained from Drosophila fly heads. Here we present a preliminary report of these studies.

Studies on ribosome structure and interactions near the m62Am62A sequence.

Antibodies raised against N6, N6-dimethyl adenosine were used to study the environment and role of the m62Am62A sequences in the E. coli ribosome. It is observed that this sequence is exposed on the surface of isolated 30S subunits, but becomes inaccessible for IgG interaction upon heat activation. The m62Am62A sequence is also inaccessible for IgG interaction in 70S ribosomes or 30S subunits immediately after dissociation of 70S particles. The presence of IgGs results in a significant inhibition of IF3 binding to unactivated 30S particles. IF3 binding to activated 30S subunits is unaffected by the IgGs. Crosslinking of 30S proteins S18 and S21 with the bifunctional phenylene dimaleimide reagents results in a reduction in the extent of 30S-IgG interaction. From what is already known about the location of S18, S21 and the IF3 binding site, it is suggested that the m62Am62A sequence is located close to the initiator tRNA binding site of the 30S subunit during initiation of protein synthesis.

Immunoglobulin heavy chain class switch from IgM to IgG in a hybridoma.

A subclone of an IgM-producing hybridoma has been identified which has switched to producing an IgG1 antibody. The parent hybridoma, PC-140, produces an antibody which binds phosphorylcholine and reacts with monoclonal antibodies that recognize myelomas of the T-15 idiotype. The IgG1 antibody binds phosphorylcholine with the same affinity as the parental IgM and also reacts with the anti-T-15 monoclonal antibodies. While the IgM-producing parent hybridoma does not express detectable surface IgM, the IgG1-producing subclone produces both membrane and secreted IgG1.

Hybridomas as a source of antibodies.

Hybridomas, the progeny of fusion between antibody-secreting spleen cells and myeloma cells, produce large quantities of a single species of antibody. They are an ideal source of serologic reagents since they can be grown in tissue culture, frozen, stored, and recovered when needed. Impending applications include production of monoclonal antibodies to a wide variety of serum components and pathogens.

Monoclonal antibodies.

Serologic reagents have played an important role in the diagnosis, treatment, and epidemiology of infectious diseases. A new technology has been developed for generating homogeneous antibodies that can be produced in large amounts and are available indefinitely. This technique promises to increase the reliability and sensitivity of immunoassays and may even provide antibodies that can be used safely in vivo.

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.