An effective antitumor immunity depends on the activation of both arms of the immune system, humoral and cellular. The ability to generate potent T cell-mediated responses against self antigens is a prerequisite for the validation of cancer vaccines in humans. While a number of potential tumor-associated antigens (TAA) have been reported in literature, the clinical applicability of many of these antigens are often not properly followed with specific immunological studies. Advances in TAA-based immunotherapy were hampered by absence of reliable animal models and inefficient vaccine approaches, in particular, poor adjuvants and delivery systems. TAA-induced immune responses often are suppressed by T regulatory cells (Tregs), a key cell type that enables evasion from anti-tumor responses.
The specific aims of our current research include: (1) the development of cancer vaccines which target/deliver antigens to various subsets of antigen presenting cells (APCs), specifically immature DCs; (2) the development of chemotoxins (chemokines fused with toxins) for the preferential depletion or modulation of ?bad? cells resulting in the enhancement of anti-cancer responses; (3) understanding the suppressive and regulatory mechanisms of T regulatory cells (Tregs); and (4) the development of novel chemoattractant peptide-based vaccine carriers and their immunomodulatory functions.? ? (1) The ideal vaccine carrier should deliver the target antigen to APCs, while, at the same time, promoting their activation. In this respect, the chemoattractant peptides appear to fulfill these requirements, in that they can both target and attract APC, and in some cases (e.g., murine beta-defensin 2) can activate cells via TLR-4. We have reported that non-immunogenic antigens were rendered immunogenic by targeting them to APCs via chemokine receptors. In vivo studies have suggested that, in order to be effective, vaccines require the targeting of immature, but not mature DCs. Recently, we have ? demonstrated that antigens delivered to chemokine receptors are efficiently internalized, processed and presented utilizing both MHC class I and II processing and presentation pathways. The chemokine-fused antigens were carried to early/late endosomal and lysosomal compartments through a clathrin-dependent process and subsequently delivered them to the cytosol for proteasomal degradation, thus facilitating efficient cross-presentation through TAP-1-dependent processes. Typically, approaches that target various endocytic cell surface receptors are known to increase the efficiency of antigen presentation by 100- to 10,000-fold. In this respect, chemokine receptor targeting is also quite efficient in that only nmol (ng/ml) quantities of tumor antigens fused with chemokine are required to mount an effective response. Therefore, this is a very simple and potent approach by which to elicit reproducibly protective and therapeutic antitumor immunity in several syngeneic murine tumor models. Recently, we have tested several novel tumor antigens to develop universal formulations for treatment of several malignant diseases. For example, we demonstrated that the embryonic antigen, OFA-iLRP, a highly conserved the ?37-kDa oncofetal immature laminin receptor? and is highly expressed in a number of human malignancies, elicited CD8+ T cell-mediated therapeutic antitumor immunity against syngeneic mouse tumors, supporting the potential application of these simple vaccines as preventive and therapeutic formulations for human use. ? ? (2). Chemokine receptors are also expressed on human malignancies, including T cell lymphoma and adult T-cell leukemia/lymphoma (ATLL). Based on this expression, we focused our efforts on developing a strategy to specifically deplete chemokine receptor-bearing malignant cells. To achieve this, we produced chimeric proteins, designated as ?chemotoxins? (chemokine + toxin), which exist as inert moieties unless delivered to the cytosol of cells. We demonstrated that CCR4-targeting chemotoxins were able to efficiently kill human T-lymphoblastoid CCRF-CEM cells (CEM) in vitro within 1-2 days of treatment (IC50 of 3-8 nM), and eradicate s.c. established CEM tumors in NOD-SCID mice. While mock treatment or the irrelevant TARC-OFA injections failed to protect mice, 100% of mice treated with CCR4-targeting chemotoxin survived and failed to demonstrate palpable tumor during the observation period. We believe that chemotoxins used alone or in combination with other modalities may enable eradication of human T cell malignant diseases when a patient?s immune system is severely immunocompromised by disease or age.? ? (3) Tregs are thought to participate in tumor survival and suppression of vaccine-induced responses. To date, precise nature of Tregs and existence of different subsets of Tregs remain largely unknown. Characterization of Tregs has been complicated by the lack of unique markers as many Treg markers are also expressed on other cell types including activated T cells. Tregs migration, particularly skin-homing, is controlled by chemokines. Using chemotoxins that are specific to various chemokine receptors, we were able to demonstrate that Tregs actually consist of CCR4+ Tregs and CCR4- Tregs. These cells differ not only by their cell surface phenotype, but also by their functional properties. While CCR4+ Tregs appear to constitutively exert suppressive activity, CCR4- Tregs (possibly as an adaptive or naive natural Tregs) require TCR-mediated activation to become fully active Tregs. Tregs utilize Fas/FasL (CD95/CD95L) signaling to mediate their immunosuppressive effects. Thus, understanding Tregs biology, particularly the role of CCR4+ Tregs, should reveal their in vivo biological relevance and their possible utilization in the treatment of cancer and various disease states. ? ? (4) An important part of our research is also dedicated to the development of ?more efficient, potent and safer? carriers that would deliver weakly-immunogenic antigens to APCs to render them immunogenic. We have explored the use of viral-derived chemokines as carriers to circumvent vaccine-induced anti-host chemokine immune responses. Our studies have revealed that viral chemokines can be efficiently utilized for vaccine delivery. Moreover, we examined a novel and as-of-yet uncharacterized function of the antimicrobial peptides, such as β-defensin-like hypothetical peptide EP2c. We believe that EP2c will possess immunomodulatory functions, as it binds APC through an unidentified receptor. This work was initiated after our study describing that murine beta-defensin 2 (mDF2b) not only recruits immature DCs through the use of the chemokine receptor CCR6, but also activates their maturation through the TLR-4. To date, none of characterized human beta-defensins were capable of activating DCs, and it is unclear whether a functional homolog of mDF2b is present among more than 30 hypothetical human beta-defensin genes reported. We have serendipitously found that mDF2b may also play a role in the elimination of activated APCs. Moreover, we also found that mDF2b also promotes a caspase-independent, TLR4/MyD88-dependent signaling cascade resulting in TNF/TNFR2-mediated apoptosis and necrosis of APCs.? ? Overall, the use of chemoattractants as vaccine carriers is an efficient and simple strategy to elicit antitumor and vaccine responses. Taking advantage of chemokine redundancy, we also demonstrated that viral chemokine fusions were equally potent in inducing protective immunity in vivo, providing a possible strategy to circumvent hypothetical, vaccine-induced anti-host chemokine autoimmunity. Moreover, we have developed a novel strategy for depletion of unwanted subsets of cells, at will, using chemotoxins. This approach should also enable us to characterize two functionally different subsets of Tregs.
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