B-cell lymphoma idiotypes chimerized by gene targeting can induce tumor immunity.

Immunization with modified immunoglobulin (Ig) idiotypes (Ids) of B-cell lymphomas is an attractive approach of experimental tumor immunotherapy. We show here that B-lymphoma cells can be gene-modified by homologous recombination at the Ig heavy chain locus. Although it has been demonstrated previously that a protein vaccine containing a mouse/human chimeric Ig had no immunostimulatory effect, we show that a xenogeneic Fc segment attached to the Id by gene targeting in autologous murine tumor cells can serve as an immunogenic carrier and is capable of inducing tumor protection. A prerequisite for successful vaccination is the delivery of tumor cells that have been engineered to express the Id in the chimeric form rather than administration of the soluble chimeric protein. Also DNA vaccination with plasmids encoding chimeric Ids was reported to induce an anti-idiotypic response, suggesting that there might be related mechanisms such as enhanced antigen presentation. Immunization with engineered lymphoma cells is a very potent protocol: in the cell-based setting, minute levels of expression in the gene-targeted cells are sufficient to confer tumor immunity. Because the titers of anti-Id antibodies induced do not reflect the degree of tumor protection, the immune mechanisms responsible for tumor rejection cannot be ascribed exclusively to a humoral response.

Induction of tumor immunity by autologous B lymphoma cells expressing a genetically engineered idiotype.

A fusion protein containing a B cell lymphoma idiotype (Id) and granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent stimulator of tumor immunity. In three different tumor models we show that immunization with autologous lymphoma cells that have been engineered to express the Id in the context of GM-CSF is much more effective than immunization with an equivalent dose of the purified protein. The lymphoma Id could be modified by introducing the GM-CSF gene into the immunoglobulin (Ig) heavy chain locus via gene targeting. This approach circumvents the isolation of the rearranged immunoglobulin variable genes from the tumor and the preparation of tumor-specific vector constructs. The low production of Id/GM-CSF fusion proteins by transfected cells, which is a major obstacle in the use of purified fusion proteins for immunotherapy, is due to the presence of the cytokine gene in the immunoglobulin locus. Low production, however, is not limiting in the cell-based setting, because upon in vivo administration of the modified autologous cells, even minute expression levels are sufficient to induce tumor immunity.

Gene therapy of B-cell lymphoma with cytokine gene-modified trioma cells.

The trioma approach is a new immunotherapeutic strategy for treating B-cell lymphomas. It is based on converting the tumour idiotype to a bispecific immunoglobulin that redirects the idiotype to antigen-presenting cells. We show here that even pre-existing tumours can be eradicated by trioma vaccination, that the trioma approach is superior to vaccination with cytokine gene-modified autologous tumour cells and that there is a synergism between trioma immunisation and GM-CSF gene transfer. Furthermore, we show that the immunising potential of GM-CSF gene-modified autologous lymphoma cells is not as dependent on the cytokine expression level as described for other tumour models, such that even minute expression rates are effective. IL-4 gene transfer in the lymphoma model is considerably less efficient or even ineffective when more sensitive systems are used. Remarkably, trioma-mediated effects are extinguished when IL-4 is expressed by the trioma cell.

Genetic stability of gene-targeted immunoglobulin loci. II. Influence of the cell line and the vector linearization site.

The site-specific integration of exogenous gene fragments by homologous recombination provides a convenient method for altering the immunoglobulin loci of B cells and specifically designing antibody molecules. To introduce a human isotype into the heavy chain locus of mouse hybridoma cells we compared the recombination frequencies of vectors that could be linearized either as integration or as replacement constructs in different cell lines. Integration as well as replacement recombination was observed, irrespective of the location of the site at which the vector was cleaved. Integration events involving the human IgG1 vectors were lost at high frequency due to secondary vector excision, so that all stable recombinations were found to be replacement events. Replacement recombination of an integration vector involves an illegitimate crossover at least at the 3' side and sometimes gives rise to deletion of the CH1 domain. However, a homologous event at the 3' side is more efficient than an illegitimate one, so that a homology that is distributed on both sides of the heterologous region promotes targeting at higher frequency than a contiguous sequence of the same total length. The position of the linearization site in the vector markedly influenced the targeting efficiency, but surprisingly, whether a double-strand break in the homology or in the heterology region more efficiently promoted integration was dependent on the cell line. In all cells, however, cleavage of the vector outside the homology region favoured stable replacements with a bias against CH1-truncated clones. We further show that the frequency of replacements induced by integration vectors is not correlated to the homology length and cannot be increased by irradiation of the cells. Our findings indicate that for targeting the IgH locus other mechanisms might be involved than at other loci.

Trioma-based vaccination against B-cell lymphoma confers long-lasting tumor immunity.

A major goal of tumor immunotherapy is the induction of a systemic immune response against tumor antigens such as the tumor-specific immunoglobulin idiotype (Id) expressed by lymphomas of the B-cell lineage. We describe an approach based on specific redirection of the tumor Id toward professional antigen-presenting cells (APCs), thereby overcoming the inefficient presentation on the parental transformed B cell. Lymphoma cells are fused to a xenogeneic hybridoma cell line that secretes an antibody against a surface molecule on APCs. Due to preferential assembly between heavy and light chains of antibodies of different species-origin, the resulting "trioma" cells produce at high yield a bispecific antibody containing the lymphoma Id and the APC-binding arm, which redirects the Id to APCs. Processing and presentation of the Id will lead to T-cell activation. An absolute requirement for inducing a complete tumor protection was the immunization with antibody-secreting trioma cells as a cell-based vaccine instead of the soluble bispecific antibody. Tumor immunity was specific and long-lasting. Both CD4+ and CD8+ T cells were necessary for inducing tumor immunity.

Genetic stability of gene targeted immunoglobulin loci. I. Heavy chain isotype exchange induced by a universal gene replacement vector.

Gene targeting at the immunoglobulin loci of B cells is an efficient tool for studying immunoglobulin expression or generating chimeric antibodies. We have shown that vector integration induced by human immunoglobulin G1 (IgG1) insertion vectors results in subsequent vector excision mediated by the duplicated target sequence, whereas replacement events which could be induced by the same constructs remain stable. We could demonstrate that the distribution of the vector homology strongly influences the genetic stability obtained. To this end we developed a novel type of a heavy chain replacement vector making use of the heavy chain class switch recombination sequence. Despite the presence of a two-sided homology this construct is universally applicable irrespective of the constant gene region utilized by the B cell. In comparison to an integration vector the frequency of stable incorporation was strongly increased, but we still observed vector excision, although at a markedly reduced rate. The latter events even occurred with circular constructs. Linearization of the construct at various sites and the comparison with an integration vector that carries the identical homology sequence, but differs in the distribution of homology, revealed the following features of homologous recombination of immunoglobulin genes: (i) the integration frequency is only determined by the length of the homology flank where the cross-over takes place; (ii) a 5' flank that does not meet the minimum requirement of homology length cannot be complemented by a sufficient 3' flank; (iii) free vector ends play a role for integration as well as for replacement targeting; (iv) truncating recombination events are suppressed in the presence of two flanks. Furthermore, we show that the switch region that was used as 3' flank is non-functional in an inverted orientation.