Current therapies for cancer, such as surgery, chemotherapy, and radiation therapy, rarely result in long-term benefits in patients with metastatic disease. Cancer immunotherapy provides an alternative approach for the control of cancer. The ideal cancer therapy should have the potency to eradicate systemic tumors at multiple sites in the body, as well as the specificity to discriminate between malignant and normal cells. In both of these respects, immunotherapy is an attractive candidate.
Recently, several studies have focused on the mechanisms of tumor escape from immune surveillance and attack that may blunt the efficacious effects of active immunotherapy. These studies have identified many distinct loss-of-function and gain-of-function mechanisms of tumor immune evasion. For example, tumors can down-modulate multiple components of the MHC I antigen processing pathway to avoid recognition by tumor-specific CTL. In addition to antigen loss, down-modulation of proteosome subunits, transporter associated with antigen presentation, β2 microglobulin, and MHC I heavy chain can diminish presentation of MHC-peptide complexes on the tumor surface. In addition, transport of MHC-peptide complexes from endoplasmic reticulum (ER) through the Golgi to the cell membrane can be diminished (for review, see ref. 1
Alternately, increased expression of a several molecules has been shown to mediate important mechanisms of tumor evasion. For example, expression of signal transducer and activator of transcription 3 (Stat3) by tumors has been identified as a central mediator of oncogenesis, angiogenesis, and immunosuppression. Recent studies have shown that Stat3 signaling in tumor cells promotes secretion of the immunosuppressive cytokines transforming growth factor-β (TGF-β) and interleukin (IL)-10, which inhibit the proliferation and function of tumor-specific cytotoxic T cells and are crucial in the accumulation of regulatory T cells in the tumor microenvironment (for review, see ref. 2
Other important tumor immune evasion mechanisms are mediated by B7-H1 (3
), indoleamine 2,3-dioxygenase (IDO) enzyme (4
), and galectin-1 (5
), as well as shedding of the natural killer cell receptors MIC-A and MIC-B (6
), which affect the proliferation, survival, or function of effector T cells. Together, these studies have laid the foundation for understanding how tumors escape immune attack and interact with the immune system in general. However, it is clear that a better understanding of the molecular mechanisms that allow tumors to escape immune attack could have a major effect on small molecule and vaccine development strategies for cancer treatment.
Most of the studies involving tumor immune evasion mentioned above have followed an approach that involves the initial identification of a new immunoregulatory molecule that is thought to play a role in tumor evasion. The next step would be to show the dysregulation of this molecule in several cancers and finally provide evidence that the molecule plays a role in the impairment of the immune response against tumors. Several molecules have been identified in this fashion, including B7-H1, IDO, galectin-1, Stat3, TGF-β, and MIC-A/MIC-B.
Recently, we took a different approach to define immune escape molecules through the in vivo
selection and characterization of an immune escape-resistant cancer cell line (7
). We developed previously a human papillomavirus-16 (HPV-16) E7-expressing cancer cell line called TC-1/P0 as a mouse tumor model for testing the E7-specific cancer immunotherapy (8
). In addition, we generated a HPV-16 E7-expressing vaccinia vaccine termed Sig/E7/lysosome-associated membrane protein-1 (LAMP-1), which encodes a fusion protein consisting of an ER signal sequence, HPV-16 E7, and the transmembrane and cytoplasmic domains of LAMP-1 to enhance antigen presentation and the development of cellular immunity (9
). Vaccination with Sig/E7/LAMP-1 vaccinia led to a substantial increase in both E7-specific CD8+
T-cell immune responses compared with wild-type E7 vaccinia, preventing the growth of TC-1/P0 in 60% to 80% of immunized mice (8
A tumor from one of the immunized mice exhibiting tumor growth was explanted and expanded in vitro. This escape variant cell line was designated P1 and injected into a new group of mice immunized with Sig/E7/LAMP-1 vaccinia. Again, a tumor from one of the immunized mice with tumor growth was explanted and expanded in vitro. This cell line was designated P2. These repeated injections with tumor cell lines allowed us to carry out in vivo immune selection and resulted in increasing resistance to prophylactic Sig/E7/LAMP-1 vaccinia immunization. After three rounds of in vivo immune selection, we obtained the P3 cell line, which was completely resistant to the vaccinia-induced immune response. Both the P0 and P3 cell lines grew with similar growth kinetics. However, when these cell lines were injected into mice immunized with Sig/E7/LAMP-1 vaccinia, P3 was able to develop into a palpable mass in all of the mice within 7 days, whereas P0 only developed in two out of five mice after several weeks. Thus, we successfully generated an immune-resistant tumor model (P3), thereby developing a system that allows us to identify genes that may contribute to tumor escape from immune attack.
Using this system, we did a functional screening assay for the gene(s) responsible for this immune-resistant phenotype. Micro-array analysis of P3 and P0 tumors revealed that vascular cell adhesion molecule-1 (VCAM-1) is one of the highly up-regulated genes expressed in the P3 immune-resistant variant compared with P0. VCAM-1, also known as CD106, is a molecule with a well-characterized role in the human immune system. It contains six or seven immunoglobulin domains and is expressed by many different cell types, including activated endothelial cells, bone marrow stromal cells, spleen stromal cells, thymic epithelial cells, peripheral lymph node (LN) and mesenteric LN high endothelial venules, and some dendritic cells in the spleen. Up-regulation of VCAM-1 in endothelial cells is induced by the cytokines IL-1β, IL-4, tumor necrosis factor-α, and IFN-γ. VCAM-1 is an endothelial ligand for very late antigen-1 (VLA-4; or α4
integrin) and α4
integrin. The interaction between VCAM-1 and VLA-4 or α4
integrin, expressed on leukocytes, is thought to be involved in the extravasation of leukocytes through the endothelium to sites of inflammation (for review, see refs. 10