Given that the general question of whether it is possible to immunize against idiotype has been answered by the completed Phase II trial and that a randomized, controlled Phase III clinical trial has been designed to answer the question of clinical efficacy, a third major research objective is to streamline the production of these individualized vaccines to make this therapy more practical. Accordingly, we have taken advantage of novel technologies and vaccine delivery systems to design alternative methods for formulating idiotype, an otherwise nonimmunogenic antigen, into an immunogenic vaccine and tested them in preclinical, syngeneic murine lymphoma models. Any delivery system which does not require protein expression holds tremendous potential for the goal of streamlining vaccine production. In our view, the most appealing aspect of DNA vaccination is its simplicity and ease of vaccine generation, although limited potency may be a limitation. In vivo expression of foreign genes encoding the tumor antigen by DNA vaccination requires only that the gene is cloned under regulatory elements of eukaryotic or viral elements into an expression cassette, which is then either injected in solution via intramuscular or intradermal routes of administration or delivered into the epidermis by particle mediated bombardment of DNA-coated gold particles (gene gun). In particular, advances in antibody engineering have made it possible to readily identify and clone both murine and human Ig variable region genes, including specific V-genes from B-cell malignancies. Once V-genes are cloned, they can be combined into a single chain Fv (sFv) format, encoding a single polypeptide consisting solely of VH and VL genes linked together inframe with a short, 15 amino acid linker. Chemotactic cytokines, termed chemokines, are thought to be among the key effector molecules regulating the trafficking of professional APC, including DC, selectively through peripheral tisues to reach lymph nodes. Chemokines are a group of small secreted proteins (7-15 kDa) that induce inflammatory responses by orchestrating the selective migration, diapedesis and activation of bloodborne leukocytes. Several chemokines, such as monocyte chemotactic protein (MCP-3), macrophage inflammatory protein (MIP)-1 alpha, macrophage-derived chemokine (MDC) and stromal cell-derived factor (SDF)-1 have been reported to be chemotactic for DC. Chemokines act by binding to a specific cell-surface heptahelical G-protein-coupled receptor, which is internalized after binding with the ligand. DC express a variety of chemokine receptors, including CCR1, CCR2, CCR5, CXCR1, and CXCR3. We explored a novel hypothesis, that the efficiency of DNA vaccination in vivo could be greatly increased by encoding a fusion protein consisting of lymphoma idiotype (sFv) fused to a proinflammatory chemokine moiety (as a substitute for KLH). The hypothesis is that anti-tumor immunity can be triggered by targeting APC in vivo with a fusion protein consisting of chemokine and tumor antigen (Nat Biotech 17:253-258, 1999). Specifically, the idea is that the expressed sFv-chemokine protein will be targeted to APC for chemokine receptor-mediated binding, uptake, and processing of sFv antigen for subsequent presentation to CD4+ and/or CD8+ T cells. The strategy has been tested on two different B-cell lymphomas, 38C-13 and A20, which express surface IgM and IgG2a, respectively. The respective sFv were cloned by RT/PCR as fusions to pro-inflammatory chemokine genes MCP-3 and IP-10, as prototypes. Chemokine fusions retained biologic function and could bind chemokine receptors and induce chemotaxis both in vitro and in vivo. These fusions have been tested as protein and DNA vaccines in both tumor models. Specifically, mice immunized by gene gun with plasids encoding IP-10- or MCP-3-sFv fusions, but not sFv alone, induced protective anti-tumor immunity against a large tumor challenge (20 times the minimum lethal dose). Moreover, T-cell subset depletion experiments revealed that MCP-3-sFv fusions induced effector CD4+ and CD8+ T cells, which were required for the protection. Furthermore, the level of protection was equivalent to that of the prototype Id-KLH protein in both tumor models, and may have been superior in the A20 model. These latter two features distinguish these sFv-chemokine fusions from other reported DNA Id vaccines. In particular, the generation of CD8+ T-cell immunity distinguishes these fusions from previously reported fusions of lymphoma Id with GM-CSF which elicited exclusively antibody responses. Another recent study reported that vaccination with DNA encoding sFv fused with fragment C of tetanus toxin elicited a CD4+ T-cell-mediated protective immune response. However, evidence for the induction of CD8+ T cells could not be found.
