[Antibody Engineering: From the Idea to Its Implementation].

The late 1970s brought opportunities to create proteins with new properties and, in particular, various derivatives of mouse monoclonal antibodies (mAbs) owing to the discoveries in molecular and cell biology and the development of bioengineering. Studies of mouse/human "chimeric" antibodies, miniantibodies to be synthesized in bacterial cells, full-size single-chain antibodies, complexes of miniantibodies with intramolecular chaperones, and other approaches made it possible to create a multitude of multifunctional biopreparations with predefined properties. The review describes, with the example of one research team, how studies in the field began and what the basis for their progress was.

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells.

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.

Expression of the chimeric IgE gene in cell culture and in various mouse tissues.

Transient expression of recombinant gene constructs is now more widely used in gene therapy as well as in DNA vaccination. In this study, the ability of one and the same genetic construct to drive gene expression both in cell culture and in tissues of the whole organism was demonstrated. Chinese hamster ovarian cells (CHO) were transfected in vitro with plasmids bearing the genes for chimeric IgE (mouse/human) antibodies under control of the human cytomegalovirus (hCMV) promoter. Secretion of recombinant IgE antibodies by transfected cells reached 60% of the intracellular concentration of antibodies. The same gene constructs were introduced into various mouse tissues using ballistic transfection in vivo. The IgE content in blood after transfection of cartilage was found to be several times lower than after transfection of the liver, spleen, or foot pad. At the same time, the content of antibodies to the xenogenous determinants of IgE was essentially independent of the tissue type. These data can be employed in selecting conditions for genetic immunization and gene therapy.

Tissue-specific isoforms of the ubiquitous transcription factor Oct-1.

The ubiquitously expressed transcription factor Oct-1 is a member of the POU protein family. It is involved in the activation of snRNA promoters and some mRNA promoters (e.g., promoters and enhancers of genes for histone H2B and immunoglobulins). In this work we have cloned and sequenced a new Oct-1 isoform, named Oct-1L. Both Oct-1L mRNA and Oct-1R mRNA (cloned earlier) are expressed in lymphocytes, but not in any other cell line tested. This is the first report of tissue-specific Oct-1 gene expression. Both these forms differ from the ubiquitously expressed Oct-1 isoforms in the N-termini. They are probably generated by alternative splicing and/or alternative initiation of transcription. The latter is confirmed by the localization of transcription start points upstream of exons 1L (lymphocyte-specific) and IU (ubiquitously expressed). We assume that tissue-specific expression of Oct-1L and Oct-1R in lymphocytes and their structural differences from the ubiquitously expressed Oct-1 isoforms may be related to B and T cell differentiation and/or expression of the immunoglobulin genes.

Oct-1 promoter region contains octamer sites and TAAT motifs recognized by Oct proteins.

The 5'-upstream region (1.3 kb) of the gene encoding the POU domain transcription factor Oct-1 was cloned and sequenced. CAT reporter gene analysis of this region has detected a functionally active promoter. This region contains 24 TAAT-core sites, arranged in five clusters (four to six sites in one cluster); two octamer sites (ATGCAAAT) are located in the first and second clusters; in the second one the CCAAT-box adjacent to the octamer overlaps with the TAAT-core site. As shown by gel retardation assay, Oct-1, Oct-2, and some unknown proteins from myeloma cell line NS/0 interact with the TAAT-core sites of these clusters. The results suggest autoregulation of Oct-1 gene expression that may also be controlled by other POU proteins, homeodomain proteins and CCAAT trans-action factors.

Conservative Val47 residue of POU homeodomain: role in DNA recognition.

Conservative Val47 residue, located in the third recognition helix of the Oct-2 POU domain, was alternately substituted with other 19 amino acids. Affinity and specificity of interaction with oct-site ATGCAAANGA and homeo-specific site ATAANGA were determined for all mutants. The wild type protein (with Val47) has maximal affinity and specificity in POU domain interaction with octamer sequence. However, V47I mutant showed stronger interaction with homeo-specific site. The highest specificity of interaction with homeo-site was recorded for V47S mutant. We conclude that only Val47 provides sequence-specific high-affinity binding of POU proteins with octamer targets other than the homeo-specific site. It is shown also that damages caused by point mutations may be at least partially compensated by participation in the oct-site recognition of both POUh and POUs domains.

Expression of immunoglobulin genes tandem in eukaryotic cells under the control of T7 bacteriophage RNA polymerase.

A tandem of recombinant mouse/human immunoglobulin (Ig) genes was constructed and inserted into the plasmid pGEM1 under the control of T7 phage RNA polymerase promoter. Sp2/0 lymphoid cell line and Chinese Hamster Ovary (CHO) cells were used as the targets for gene transfection. Both cell lines contained in their genomes a T7 RNA polymerase gene modified with a nuclear-located signal derived from SV40 large T-antigen. Cell lines transfected with the gene tandem effectively synthesized mRNA (up to 9 x 10(3) bp) that hybridized with epsilon- and kappa-gene probes. Separate transcripts corresponding to mRNAs of individual heavy and light chains were not detected in either transfected cell line. It follows from these data that transcription in the transfected cells is controlled mainly by the T7 phage polymerase promoter. Both lymphoid and nonlymphoid cell lines transfected with the gene tandem synthesized the epsilon-heavy (70 kDa) and kappa-light (25 kDa) Ig polypeptide chains. Production of chimeric antibodies by the myeloma Sp2/0 cells was higher than that by the CHO cells. Individual clones synthesized up to 150 ng/mL chimeric IgE. However, only lymphoid Sp2/0 cells were capable of efficient secretion of the recombinant antibodies. The mechanism of translation of mRNA synthesized in eukaryotic cells by T7 phage RNA polymerase is discussed.

Production of recombinant antibodies in lymphoid and non-lymphoid cells.

A recombinant tandem of 'chimeric' mouse/human immunoglobulin (Ig) genes was constructed and inserted into plasmid pGEM1 under the control of the T7 bacteriophage RNA polymerase promoter. Lymphoid (Sp2/0) and non-lymphoid (CHO) cell lines used for transfection contained in their genomes a semisynthetic gene of T7 RNA polymerase and steadily expressed this enzyme. It was shown for the first time that a stable polycystronic transcription of the Ig gene tandem occurs under the control of a single T7 phage promoter, both in lymphoid and non-lymphoid cells. Synthesis of kappa-light and epsilon-heavy Ig chains and functionally active antibodies was observed in the above-mentioned transfected cell lines.

Allelic variants of rearranged immunoglobulin heavy and light chain genes in hybridoma PTF-02 and parent myeloma.

The hybridoma PTF-02 secretes an antibody against pig transferrin. Rearranged genes for heavy and light immunoglobulin chains have been studied in the genomes of this hybridoma and in the parent myeloma P3-X63.Ag8.653. The hybridoma was shown to contain three rearranged allelic variants of the heavy chain gene's locus. The gene H2, responsible for synthesis of the heavy chain of the antibody to transferrin, was transmitted in the hybridoma cell from a lymphocyte. Two other genes (H1 and H3) were found both in the hybridoma and parent myeloma genomes. The gene H1 was identified in MOPC21 myeloma, which is a precursor of the X63.Ag8 descendent line. Rearranged k genes were also identified both in the hybridoma and parent myeloma. A functional (K2) gene and a fetal (F) gene appeared in the hybridoma genome from an antigen-stimulated normal lymphocyte. The fetal gene was lost in the course of continuous cultivation of the hybridoma PTF-02 cell line. The gene K1 was transmitted from the myeloma used for fusion. In such a way, the pedigree of rearranged heavy and light chain genes in the hybridoma PTF-02 was established. The results obtained in this work may be relevant to many hybridomas whose immortalizing fusion partner is a MOPC21 derivative, and allow one to identify and isolate functional variable genes to create recombinant constructions.