In this study, we have demonstrated that transgenic expression of CCR2b on ex vivo expanded ATCs dramatically enhances both in vitro and in vivo chemotaxis in response to CCL2 produced by neuroblastoma cell lines and primary tumor cells. When the activated T cells also express a chimeric antigen receptor directed to the tumor associated antigen GD2, co-expression of CCR2b leads to rapid migration to a subcutaneous neuroblastoma xenograft associated with greater anti-tumor activity than CAR expressing ATCs that lack CCR2b
labeling and in vivo tracking of ATCs expressing a CAR specific for the α-folate receptor showed that these cells retained the capacity to migrate to ovarian cancer after non-specific activation and expansion (1
). Similarly, ATCs transduced with the α and β chains of MART1 and gp100-specific T cells induced disease regression in patients with metastatic melanoma and were present as infiltrates in tumor biopsies (13
). In principle, therefore gene modified, activated and expanded T cells can continue to migrate to tumor sites, and the failure of migration observed in patients with tumors such as neuroblastoma suggests instead that their absence at tumor sites may be a consequence of a mismatch between the chemokines the tumors produce and the chemokine receptors expressed by ATC.
CCL2 is a chemokine active for CCR2 expressing T lymphocytes (14
), NKT cells (16
) and γδ T cells(17
), and our results indicate that transgenic expression of the CCR2 receptor is both necessary and sufficient to restore CCL2 responsiveness to T cell subsets in which receptor expression is deficient. Our data demonstrating an absence of CCR2 on ATCs, a lack of CCL2 responsiveness and a failure of migration to tumor sites differ from some earlier findings in which CCR2 was detected on human ATCs (2
) at a sufficient level for CCL2-induced T cell migration in tumor bearing immune deficient mice (19
). While these differences may be explained on the basis of distinct culture conditions, including the use of anti-CD28 antibody which may facilitate downregulation of CCR2 following TCR activation (20
), it is likely that the transgenic CCR2 is expressed at higher levels and on a greater percentage of cells than previously reported and will likely lead to enhanced tumor migrations even in subjects with low constitutive expression of CCR2.
More aggressive MYCN amplified tumors transcriptionally repress production of CCL2 (16
) suggesting that CCR2b modification may be of limited value in high-risk disease. However, of these high-risk patients(stage 3,4), approximately 70% lack MYCN amplification (22
). Further, while MYCN status is used for risk stratification, Matthay et al showed that in those patients receiving high dose chemotherapy followed by autologous hematopoietic stem cell rescue (AHSCT) as consolidation therapy (the current standard of care), MYCN amplification proved to be a negative prognostic factor in overall survival (OS) but not event-free survival (EFS) (23
). This indicates that a substantial number of high-risk patients with relapsed/refractory disease may ultimately benefit from CCL2-directed T cell therapies.
CCL2 has also been found to be highly secreted by the tumor microenvironment in a variety of malignancies including melanoma, sarcoma, glioma, ovarian, prostate and breast cancer (24
). CCL2 production is associated with increased infiltrating tumor-associated macrophage (TAM) that promote tumor growth via cytokines such as IL-6 (30
), enhance angiogenesis (31
), and contribute to immune suppression through production of IL-10 (34
) and recruitment of Th2 and regulatory T cells (35
). Moreover, CCL2 is produced by TAMs themselves, suggesting a self-amplifying loop(37
). The abundance of CCL2 in solid tumors, produced both by tumor cells and infiltrating immune cells, implies that this chemokine pathway might be used as a generic mechanism to induce tumor-specific migration of cytotoxic T cells for cancer treatment, irrespective of whether CCL2 is produced by the tumor cells themselves.
Under physiological or stress conditions, CCL2 is secreted by non-malignant cells including endothelium, fibroblasts, smooth muscle, and astrocytic and microglial cells(38
). CCR2b-modified T cells may therefore migrate to these tissues resulting in decreased antitumor effects or unintended toxicity. The amounts and concentration of CCL2 produced at tumor sites, however, make it probable that the bulk of effector cells will go to the tumor site. The use of autologous virus-specific T cells transduced with the GD2-CAR may further reduce the potential for off-target toxicity against non-tumor tissues (4
In summary, using neuroblastoma as a model, we show that transgenic expression of a chemokine receptor complementary to tumor-associated chemokines will enhance the ability of ATCs to traffic to tumors and improves their anti-tumor efficacy. In principle, this approach should be applicable to any tumor directed T cell lacking a receptor that matches the chemokines the tumor produces.