Ovarian cancer chemokines may not be a significant barrier during whole tumor antigen dendritic-cell vaccine and adoptive T-cell immunotherapy

ABSTRACT

Tumor barriers preventing T-cell homing and engraftment should be neutralized during cancer immunotherapy. We recently discovered that ovarian cancer expresses quasi-universal chemokines that can support T-cell homing. Furthermore, T cells elicited by whole tumor antigen dendritic-cell vaccines express cognate chemokine receptors which are upregulated by CD3/CD28 costimulation.

Recent therapeutic advances in ovarian cancer have failed to significantly improve the survival of patients diagnosed with advanced stage disease. Ovarian cancer is characterized by preexisting immune circuits that can either favor patient survival, in which case the tumor is infiltrated by intraepithelial effector T cells,¹ or predict poor prognosis among patients with tumors infiltrated by immunosuppressive regulatory T cells (Tregs),² B7-H4 expressing macrophages² or vascular endothelial growth factor (VEGF) overexpressing tumor and stromal cells.¹ As adaptive immune T-cell responses against a myriad of tumor-associated antigens (e.g., NY-ESO-1, p53, Her2/neu, WT1, and MAGE3) have been documented, ovarian cancer is considered an immunogenic tumor and represents a good candidate for immunotherapy.

The success of immunotherapy strongly depends on the ability of T cells to home to the tumor microenvironment. This complex process requires adhesion and extravasation of T cells through endothelial cells (ECs), a crucial step often inhibited by the tumor vasculature itself. Tumor ECs, under the influence of VEGF and other paracrine mediators that control inflammation such as interleukin 10 (IL-10) or prostaglandin E₂ (PGE₂), establish a molecular and cellular barrier limiting T-cell infiltration.³ VEGF deregulates vascular cell adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) on ECs, thus impeding T-cell adhesion and extravasation, whereas VEGF, IL-10 and PGE₂ upregulate Fas ligand on tumor ECs, thereby inducing apoptosis in transmigrating Fas-expressing T cells.⁴ Thus, the vascular barrier plays an important role in controlling tumor immune infiltrates. Importantly, pharmacologic suppression of these pathways can increase T-cell trafficking to the tumor microenvironment,⁴ hence enhancing immunotherapies through combinatorial approaches.

Besides the vascular barrier, chemokine networks in tumors also play a key role in shaping immune cell tumor infiltration and could also represent a blockade to immunotherapy, particularly if the chemokine milieu is not conducive to T-cell homing and engraftment. Chemokines and their receptors constitute master regulators of homing and retention of immune cells to target sites. Additionally, certain chemokines exert multifaceted roles as they promote cancer cell proliferation, invasion and tumor angiogenesis.⁵ Thus, the tumor microenvironment may naturally polarize the tumor microenvironment toward a chemokine milieu that augments tumor growth and immune escape.

To shed light into the unexplored ovarian cancer chemokine networks, we mapped the chemokine landscape of advanced stage serous ovarian cancer using 3 large separate patient cohorts that shared the same clinical and biological characteristics as well as 13 established primary ovarian cancer cell lines.⁶ Our results demonstrated that the chemokine landscape of ovarian cancer is heterogeneous with high expression levels of known lymphocyte-recruiting chemokines. For instance, tumors with pre-existing tumor-infiltrating intraepithelial T cells expressed high levels of CCL2, CCL4, CCL5 and CCL8 in tumor islets. On the other hand, CXCL10, CXCL12 and CXCL16 were expressed quasi-universally in nearly all tumors, including tumors lacking intraepithelial T cells.⁶ Importantly, the chemokine milieu in metastatic deposits resembled the primary site, suggesting minimal heterogeneity.⁶

Immunotherapy approaches for patients with recurrent ovarian cancer remain limited. Anti-cancer vaccination using a whole tumor lysate dendritic-cell (DC) vaccine approach to multiple tumor-associated antigens has proven a useful strategy to elicit or enhance antitumor immune responses, particularly prior to adoptive cell transfer.⁷ Our group recently initiated a phase I/II clinical trial in patients with recurrent stage III/IV ovarian cancer using autologous DCs loaded with HOCl-oxidized autologous tumor lysate in combination with anti-angiogenic bevacizumab and low-dose cyclophosphamide.^8,9 Patients in which DC vaccination produced clinical benefit but still presented with evidence of disease following 3 months of therapy were offered to undergo apheresis to collect vaccine-primed peripheral blood T cells. These DC-primed, autologous T cells were subsequently CD3/CD28-costimulated and expanded ex vivo and reinfused after lymphodepleting chemotherapy in order to boost antitumor immune responses.¹⁰

To understand whether the T cells elicited by our immunotherapy approach are equipped with cognate chemokine receptors to home to ovarian cancer chemokines, we analyzed the chemokine receptor expression patterns among HER-2/neu tumor-specific T cells. We observed that DC vaccine-primed peripheral blood T cells natively expressed CCR10 (the receptor for CCL27 and CCL28), CXCR3 (the receptor for CXCL9, 10 and 11) and CXCR4 (the CXCL12 receptor), and the costimulation and expansion of these cells ex vivo significantly upregulated the expression levels of CXCR3 and CXCR4. Furthermore, we confirmed that these receptors were functional and specified T cell migration toward their cognate chemokines.⁶

The above results indicate that although many ovarian cancers do not exhibit intraepithelial T cells, they do express some chemokines within the tumor islets that could support T-cell homing and engraftment. Additional work is required to understand whether these “quasi-universal” chemokines are indeed functional and the mechanisms underlying the lack of intraepithelial T cells in these tumors despite the expression of such chemokines. It is possible that these quasi-universal chemokines are functionally impaired or that their net effect is to promote immunosuppression rather than supporting the orchestration of antitumor T-cell attack. For the time being, we have designed our clinical study to counter some of other known inhibitory mechanisms in ovarian cancer that our work has uncovered to date, including the vascular barrier and the presence of immunosuppressive Tregs. We seek to normalize the tumor vascular barrier via bevacizumab to inhibit VEGF and therapeutic aspirin to block PGE₂, whereas we aim to attenuate Tregs with low-dose cyclophosphamide. Other therapeutics strategies that may prove beneficial to effectively mobilize tumor immunity after successful vaccination may include suppression of immune checkpoints (e.g., PD-L1) or other tumor-derived soluble factors and enzymes (e.g., TGFβ, IL-10 or IDO-1). The expanding compendium of potent immunomodulatory pharmacologic agents will open the door for the design of more effective immunotherapeutics in the near future.

Figure 1.

Autologous-tumor pulsed DC vaccination combined with adoptive T-cell immunotherapy and the chemokine landscape in ovarian cancer. Ovarian tumors are surgically resected and PBMCs are collected via apheresis. Whole tumor lysate is prepared to pulse PBMCs-derived DCs and administered to the patient in a vaccine formulation. Vaccine primed T cells collected after DC-vaccination are ex vivo expanded and co-stimulated with CD3/CD28 beads and adoptively transferred to patients. Chemokine expression levels were analyzed to characterize the ovarian tumor chemokine landscape. Adoptively transferred T cells expressed and upregulated the cognate chemokine receptors and showed enhanced migration toward universally expressed chemokines in ovarian cancer.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by NCI P01-CA83638 SPORE in Ovarian Cancer, R01 FD003520, the Ovarian Cancer Research Fund, and the Ludwig Institute for Cancer Research.