Primeboost Strategies In Cancer Immunotherapy

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Immunization has traditionally relied on repeated administration of antigen to augment the magnitude of the immune response. With the advancement in recombinant DNA technology, genetically engineered vaccines such as expression plasmids, recombinant proteins, viruses, and bacteria have become the latest modalities for vaccine development. The first such vaccine in its class, a hepatitis B virus vaccine in the form of recombinant protein produced in yeast, has been shown to be potent in providing protective efficacy in humans (43,44). While this homologous protein-based immunization is very effective for generating humoral immune responses, it is generally inefficient in inducing cell-mediated immunity important for protection against infections caused by intracellular pathogens and for cancer immunotherapy. DNA vaccines, on the other hand, have been tested in small and large animal models, and have demonstrated efficacy in inducing both humoral and cellular immunity for infectious diseases and cancer, while clinical trials of such DNA vaccines have provided mixed results (45). In parallel with the development of DNA vaccines, recombinant viral vectors, such as poxviruses and adenoviruses, have emerged as vaccine delivery systems. In mice as well as nonhuman primates, recombinant viral vectors are very efficient for the induction of cellular and humoral immunity, characterized by increased CD4+ and CD8+ T cells as well as antibodies. However, DNA vaccines or recombinant viral vectors failed to induce the high levels of antigen-specific T cells necessary for protection against intracellular pathogens when used singly or with repeated administration (homologous boosting). This has led to the investigation of whether heterologous prime-boost immunizations with different modalities can elicit immune responses of greater magnitude and quality than can be achieved by priming and boosting with the same vector.

It has been well established that cellular immunity is the key in controlling tumorigenesis and microbial infections of intracellular pathogen that involves induction and expansion of antigen-specific T cells endowed with multiple capabilities such as migration, effector functions, and differentiation into memory cells. Earlier studies attempted to elucidate the sequence and combination of modalities during a heterologous prime-boost immunization regimen that results in the generation of antigen-specific memory T cells by priming followed by amplification of these cells by boosting. A variety of such vaccine components have been evaluated in preclinical models or human trials including DNA plasmid, recombinant poxviruses and adenoviruses, alphavirus replicon particles, modified vaccinia virus Ankara, and protein or peptide in adjuvants. Results from these studies revealed that multiple mechanisms account for the efficiency of prime-boost vaccination protocols; however, synergy in epitope presentation during priming and boosting by different expression vectors is the key requirement to evoke high-avidity CD8 cells in the host, while additive effect may result from other features within the boosting vector. Table 1 summarizes some major findings from such studies in humans.

DNA vaccines have been widely accepted as good priming agents since they can trigger antigen presentation via both MHC class I and class II, thereby inducing both CTL and Th1 lymphocytes via a mechanism of action depicted in Figure 1. To leverage the high quality of the immune response primed by plasmid DNA and likely facilitated by its excellent toll-like receptors 9 (TLR9)-dependent and -independent adjuvant activities, various groups have explored boosting strategies by delivery of targeted antigen incorporated in different forms. Such boosting components encompass proteins, live viral vectors, or plasmid vectors. There is an extensive database obtained in preclinical models and in clinics supporting this concept, mostly in the area of prophylactic immunization for infectious diseases targeting pathogens such as HIV (13), malaria (53), and tuberculosis (54). Priming with plasmid DNA and boosting with live vector has shown, by far, to be the most effective regimen to induce immune response at the level of therapeutic usefulness both in preclinical and in clinical trials (46,48,55,56). For cancer immunotherapy, a pioneer work a decade ago by Irvine et al. showed that heterologous prime-boost strategy can augment antitumor immunity by generating a strong antigen-specific CTL response in mice (57). Their data suggested that immunizing with DNA and boosting with a live viral vector expressing the same tumor-associated antigen prolonged the survival of tumor-bearing mice more efficiently than multiple immunizations with the same vector, correlating with stronger specific CTL responses (57). Meng et al. performed a similar study on mice by administration of plasmid DNA encoding murine a-fetoprotein followed by boosting with a nonreplicating adenoviral vector expressing the same antigen (58). This immunization strategy resulted in elicitation of high frequency of Thl-type a-fetoprotein-specific cells leading to tumor protective immunity in mice at levels comparable with a-fetoprotein-engineered DCs (58). However, in clinic, data from a phase I trial of sequential administration of plasmid DNA and adenovirus expressing L523S protein in patients with early-stage non-small-cell lung cancer showed a high level of safety but limited evidence of L523S-directed immune activation (59), suggesting that a further optimized immunization approach is needed to break immune tolerance or ignorance to self-antigens.

Despite the mounting data demonstrating the tolerability of live vectors in preventive vaccination, there is significant safety concern for the use of such vectors in cancer patients who may be at the stage of immune suppression after prolonged chemotherapy. Additional drawbacks of the use of live viral vectors include antibody responses to vectors that diminish effectiveness of later boosts, and higher development and production costs. Therefore, considerable efforts have been devoted to explore nonviral options for boosting immunity generated during the DNA-priming interval. DNA vector itself has been shown to induce suboptimal immunity even after repeated immunization. However, the use of DNA vectors with a modified sequence, or delivered in a different mechanism, as a boosting agent, lead to a significantly improved immune response. Preclinical animal models have demonstrated that the use of an "altered self' form of antigen may provide CD4+ T cell help to break the tolerance and to induce tumor protection (60). Such hypothesis is further tested in mice with a prime-boost immunization regimen with plasmids expressing human or mouse tyrosi-nase-related protein 1 (TRP-1) (60). That priming with human TRP-1 DNA broke tolerance to mouse TRP-1 was evidenced by the manifestations of auto-immunity, characterized by coat depigmentation, and such immune responses to TRP-1 provided significant protection against colonization of the lung by metastatic melanoma cells (60). The presence of slight differences in epitopes between host "self' protein and that encoded by xenogeneic DNA plasmid vaccine, along with inherent bacterial unmethylated CpG motifs, may be sufficient to boost the immune response to break tolerance and ignorance to tumors. Currently, such approaches are in clinical proof of principle testing with two well-defined tumor antigens-prostate-specific membrane antigen and tyrosinase (61,62). Another approach is priming with naked DNA and boosting with the same vector in combination with the use of an electroporation device to improve the immune responses. In both animal and clinical trials with prophylactic DNA vaccines, electroporation enhances immune responses to DNA vaccines by increasing gene expression as well as inducing inflammatory cell infiltration (63). Such strategy has also been explored in cancer with two tumor models, the CT26 carcinoma

Table 2 Advantages of Plasmid Vectors as Vaccines

Features

Induction of broad immune responses encompassing MHC class I-restricted T cells

Predominant induction of T1 immune responses Beneficial safety profile

Simplicity of manufacturing

Mechanism of action/rationale

Direct transfection of APCs and/or cross-presentation

Binding to TLRs and activation of dependent innate immune pathways Lack of replication, transient, episomal persistence, infrequent genomic integration Straightforward E. Coli fermentation process and plasmid purification

Abbreviations: APCs, antigen-presenting cells; MHC, major histocompatibility complex; TLR, toll-like receptors.

and the BCL1 lymphoma (64,65). It is interesting to note that for such homologous prime-boost approach, the most effective way to generate a potent immune response is priming with naked plasmid DNA and boosting with the same vector using an electroporation device. The mechanism of improved immune response by electroporation of vaccine plasmids during boosting is still not completely understood; however, it involves at least two features, namely, increased antigen expression and necrosis at the injection site, which induce inflammation. It is also possible that in electroporated APCs, an elevated antigen expression may also introduce a subtle shift of antigen presentation, a slight different processing compared to that of cells in vivo transfected with naked DNA, providing additional CD4+ T cell help (Table 2).

Other nonviral vectors such as the simplest ones, namely, the peptides, generally have a poor pharmacokinetics profile. Recently, it was shown that intra-LN injection of T-cell epitope peptides may actually circumvent their poor pharmacokinetics and leverage their intrinsic immune properties (66). More significantly, plasmid priming and peptide boosting achieved extremely robust immune responses (nearly 1/5 CD8+ T cells specific for a given epitope; Fig. 2) in a preclinical model consisting of immunization of HLA-A2 transgenic mice (67) immunized against Melan A antigen. Not unexpectedly, this outcome could be achieved only by intra-LN administration of both plasmid and peptide rather than subcutaneous injection, reinforcing the importance of targeted delivery for the purpose of accessing APCs. In addition, with such a high number of epitope-specific CD8 cells, it becomes feasible to delineate the functional role of vectors by defining the phenotype of specific T-cell population during priming and boosting. By using cell separation techniques, multicolor FACS analysis, and ex vivo functional evaluation, it has been demonstrated that while plasmid priming generated both central and peripheral memory T cells, peptide boosting had a

Figure 2 Comparison between the immune responses achieved by intra-LN and subcutaneous injection of plasmid and peptide in a plasmid prime-peptide boost fashion. Equivalent dosage, 25 mg of plasmid (pSEM) or peptide (Melan A 26-35 analogue) in 25 mL of sterile PBS, was administered to HHD transgenic mice via the intranodal injected and/or administrated in subcutaneous route. The number of injections remained the same. The results were expressed as percent tetramer CD8+ T cells within the CD8+ T cell population, measured in blood, at 15 days after the completion of the immunization protocol (mean ± SEM; n = five mice per group). Abbreviations: LN, lymph nodes; PBS, phosphate-buffered saline.

Figure 2 Comparison between the immune responses achieved by intra-LN and subcutaneous injection of plasmid and peptide in a plasmid prime-peptide boost fashion. Equivalent dosage, 25 mg of plasmid (pSEM) or peptide (Melan A 26-35 analogue) in 25 mL of sterile PBS, was administered to HHD transgenic mice via the intranodal injected and/or administrated in subcutaneous route. The number of injections remained the same. The results were expressed as percent tetramer CD8+ T cells within the CD8+ T cell population, measured in blood, at 15 days after the completion of the immunization protocol (mean ± SEM; n = five mice per group). Abbreviations: LN, lymph nodes; PBS, phosphate-buffered saline.

profound impact in differentiation and relative expansion of CD62L- peripheral memory CD8+ T cells. The latter was paralleled by migration of CD8+ T cells out of LNs to nonlymphoid organs, along with a gain of function for interferon (IFN)-g and chemokine expression, a key feature of peripheral memory/effector T cells. Figure 3 depicts a model of the prime-boost approach and the impact of plasmid and peptide on various T-cell subsets, respectively.

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