Versatile expression system for rapid and stable production of recombinant proteins.

Previously we reported the development of a novel expression system with Tat/TAR-oriP vectors and HKB11 cell line, which supports high level protein expression (Cho et al. Cytotechnology 2001, 37, 23-30). In the present study, we further demonstrated that HKB11 cells are suitable for high throughput expression (microgram scale) of genomic candidates in transient transfection system for in vitro evaluation of biological functions. HKB11 cells were also shown to support the production of milligram to gram quantities of protein drug candidates for in vivo evaluation of efficacy in various disease models. Stable HKB11 clones secreting high levels of a tissue factor (TF; 40-50 pg/c/d) and B-domain deleted recombinant factor VIII (BDDrFVIII; 5-10 microU/c/d) were derived under serum-free conditions. The specific productivity for these two proteins from the HKB11 cells was 10-fold greater than those from CHO cells derived under the similar conditions. In conclusion, we have demonstrated that the HKB11 cell line is well-suited for transient and long-term production of recombinant proteins.

Generation of novel killer hybridomas derived from proliferation-suppressed somatic cell hybrids between YACUT T cell lymphoma and normal lymphocytes activated in secondary mixed lymphocyte cultures.

Somatic cell hybridization between the YACUT T cell lymphoma cell line with normal lymphocytes activated in secondary mixed lymphocyte cultures (MLCs) consistently yielded IL-2-dependent CD4- CD8 alpha+ beta- Fc gamma RIII+ hybrids with cytotoxic function. The hybrids expressed T cell receptors other than that of YACUT origin, and fusion of the YACUT with a CD8 alpha+ beta+ Fc gamma RIII- T cell line also yielded hybrids with an unexpected CD8 alpha+ beta- Fc gamma RIII+ phenotype, which two observations strongly suggested that CD8+ T cells became the parental cell of the hybrids. Prolonged growth of the hybrids with IL-2 resulted in the generation of autonomously growing hybrids (hybridomas) without abrogating the cytotoxic function. The hybridomas exhibited MHC-unrestricted cytotoxicity in a Ca(2+)-dependent manner without prior stimulation and also mediated antibody-dependent cellular cytotoxicity. These results indicate that novel killer hybridomas can be produced following cell transformation of proliferation-suppressed cytotoxic YACUT x MLC cell hybrids. The killer hybridomas may be of value for analyzing recognition mechanisms and molecules involved in MHC-unrestricted cell-mediated cytotoxicity.

Generation of somatic variants of a B cell hybrid mediated by a non-cytolytic L3T4+ idiotype-specific T cell.

We previously reported that idiotype (Id)-loss, stable somatic variants of a B cell hybrid, 2C3E1, are generated both in vitro and in vivo, after interaction of the Id-positive tumor cells with autologous Id-specific effector T cells. The present investigation was undertaken to elucidate further the nature and functional characteristics of the effector T cells. We report here that the idiotype-specific cells mediating the generation of Id- tumor variants are Thy1+ L3T4+ Lyt-2- cells, which respond to specific idiotypic stimulation by secreting IL-2 in vitro. No IL-2 is secreted in response to unrelated Ig or an Id/Ig-2C3E1 tumor variant. Furthermore, the Id-specific T cells exert strong suppressive effects on the expression of 2C3E1 Ig and the effects can be reversed by blocking the L3T4+ T cells with monoclonal anti-L3T4 antibody in vitro during the initial 3 days of co-culture. After 4 days, the T cell-mediated suppression of the 2C3E1-Id is irreversible. In addition to the in vitro studies we have determined that the administration of anti-L3T4 mAb to mice just before priming with idiotype-bearing tumor cells also abrogates the suppressive effects of the idiotype primed spleen cells on Ig expression of 2C3E1. To study the Id-specific effector T cells in more detail we have generated functional Id-specific L3T4+ T cell lines. These T cell lines have been shown to recapitulate the generation of Id- tumor variants that we observed with Id-primed spleen cells. It is concluded that L3T4+, Id-specific Ts cells are responsible for the generation of somatic variants of the B cell hybrid 2C3E1 and that the induction or selection of these variants progresses from a reversible phase to an irreversible phase.

Isolation of rat/rat T cell hybrids using W/Fu C58(NT)D as fusion partner.

Functional rat/rat T cell hybrids were isolated for the first time by the fusion of spleen cells from rats tolerized to the hapten TNP to a HAT-sensitive rat thymoma (C58(NT)D). 11 fusions using different protocols were attempted to assess the optimal conditions for high hybridization frequency of the desired specificity. Interestingly, the cell density requirement of the non-transformed fusion partner took precedence over that of the C58 cell line, i.e., rat cells needed to be at high density after fusion, but others have reported that mouse cells prefer a much lower density even when the same line (C58) is used. Six fusions yielded hybridomas with between 3% and 70% of wells containing hybrids after three weeks of culture, depending on the protocol used. Phenotypically, all of the hybrids and clones tested expressed the W3/25 (rat CD4) antigen, but no OX-8 (rat CD8) or immunoglobulin molecules. A minority of hybrids were found to secrete factors able to suppress (a) proliferation, (b) antibody production, and (c) cells bearing IL-2 receptors, but none appeared to suppress the production of IL-2 itself. In contrast to non-transformed rat T cell lines, the T hybrids isolated were easy to grow to high densities, clone and freeze without the need to add exogenous antigen or lymphokines to the cultures at any stage.

Cell-interaction molecules on immunocompetent lymphocytes. Development of anti-parent cell-interaction-molecule-receptor reactions in F1 hybrid mice and evidence for a unique F1 hybrid subset of interacting cells.

The experiments presented herein demonstrate that F1-parent T-B cell cooperation in vivo is significantly diminished by the addition of lymphoid cells of opposite parental type. This inhibition phenomenon is not a straightforward allosuppression mechanism as (a) it can be induced by parental lymphoid cells depleted by T cells, (b) it does not operate on cooperative interactions between homologous T and B cells of opposite parental type, and (c) absolutely requires the presence of F1 cells as participants in the reactions generated. The possible involvement of alloantibodies produced aberrantly under the experimental conditions employed has been ruled out by direct macrophage/antigen-presenting cell components of the reactions has been excluded. Because the presence of parental lymphoid cells only affects cooperative interactions between F1 T cells and B lymphocytes of opposite parental type but has no inhibitory effect on cooperative interactions between homologous F1, T and B cells, this (and other points discussed herein) strongly argues for the existence of one or more subsets of F1 interacting partner cells that are uniquely specific for F1, as distinct from either parental type cell interaction determinants. For reasons discussed, it appears that the most likely mechanism underlying such parental cell-induced inhibitory effects on F1-parent partner cell interactions is the development of anti-self cell interaction structure responses by F1 cells against the relevant self-specific cell-interaction structures of the parental partner cells involved.

Chromosome-mediated gene transfer results in two classes of unstable transformants.

The human thymidine kinase gene has been transferred from HeLa S3 cells to mouse LM(TK-) cells via isolated metaphase chromosomes. Efficient transfer of the thymidine kinase gene (1.8 X 10(-5) colonies per recipient cell) was obtained when the donor chromosomes were precipitated with calcium phosphate and the recipient cells were treated with 10% (vol/vol) dimethyl sulfoxide. Thirty-five independent cell lines were analyzed in detail. Cytologically detectable donor chromosome fragments were observed in 14% of the cell lines. Many of the transformed cell lines were also found to express the human genes for galactokinase (23% of the transformed cell lines) and procollagen type I (69% of the transformed cell lines), which are syntenic to thymidine kinase on human chromosome 17. On the basis of stability analyses, three classes of transformed cell lines were defined and characterized. One class of transformants was stable, showing no loss of the transferred phenotype in the absence of selection. A second group of transformants was unstable, losing the thymidine kinase phenotype at a rate of 1.5-2.5% per day. This group of transformants was found to possess large donor chromosome fragments (macrotransgenomes) and relatively low levels of donor gene activity. The third group of transformants lost the thymidine kinase phenotype rapidly, at a rate of 6-10% per day. These cell lines contained small, cytologically undetectable transgenomes (microtransgenomes) and overexpressed the transferred thymidine kinase gene.