Adoptive cellular immunotherapy (ACI) treats disseminated cancers with ex-vivo expanded tumor-reactive lymphoblasts. These cells traffick poorly into target lesions contributing to the mixed results observed in therapeutic trials. This grant focuses on the mechanisms and clinical consequences of leukocyte recruitment into sites of metastic disease during ACI with vaccine-primed, ex-vivo expanded lymph node cells. New preliminary studies show that a single-dose of TNF-alpha, administered intraperitoneally 4 hours prior to infusion, increased the recruitment of infused, vaccine-primed cells into lungs with established micrometastases. This treatment promoted inhibition of tumor-growth by these cells regardless of the protocol used for ex-vivo expansion. Pretreatment with TNF-alpha alone was inactive at the concentration used (one mu g/animal, single dose) and did not produce visible symptoms. New preliminary studies also suggest that the vaccine-primed cells responsible for inhibition of tumor growth (the Tc/h-1 subsets) can be distinguished from those that suppress this response (the Tc/h-2 subsets) based on high levels of selectin-ligand expression. The PI proposed that selectin-mediated recruitment of the Tc/h-1 cells into sites of metastatic disease is essential for inhibition of tumor growth. Consequently, transient induction of selectins on the vasculature associated with metastases as well as enrichment of the Tc/h-1 subset in the population of infused cells should improve clinical outcome. Furthermore, selectin-based fractionation of vaccine-primed and expanded cells should both enrich the Tc/h-1 subsets and deplete the Tc/h-2 subsets.
Specific Aim 1 investigates the adhesion receptors required for the recruitment of tumor-draining lymph node cells (TDLN) and host leukocytes into lungs with 3-day established pulmonary micrometastases. TDLNs labeled with stable fluorescent dyes or congenic markers, receptor-specific antibodies and mice with disrupted selectin-genes will be used in these experiments. The relative contributions of TDLN and host cell recruitment to clinical outcome will also be determined.
Specific aim 2 investigates the adhesion receptor phenotype of TDLN and VPLN (human-counterparts currently in clinical trials) that secrete high levels of IFN-gamma and GM-CSF (Tc/h-1) and high levels of IL-4 and IL-10(Tc/h-2). In addition, selectin-based fractionation procedures will be developed that separate the Tc/h-1 and Tc/h-2 subsets. The fractionated populations will be compared to the starting population and each other for activity against malignant cells in vitro and in vivo.
Specific aim 3 determines whether induction of endothelial adhesion receptors on tumor-associated vessels augments TDLN recruitment and inhibits tumor-growth. The new preliminary studies cited above support for this hypothesis. Proinflammatory cytokines, such as TNF-alpha, will be administered prior to infusion of TDLNs. The impact of these preparative regimens on the behavior of standard and fractionated TDLN (Tc/Th-1 and Tc/Th-2 subsets; high and low selectin-ligand expression) will be established. The impact on TDLN activity against poorly immunogenic or large neoplasms, on the number of cells required for inhibition of metastases and on the dose or duration of systemic IL-2 therapy will be assessed. The role of adhesion receptors and recruitment (TDLN and host cells) will then be determined for preparative regimens that improve clinical effectiveness. The extensively revised grant rectifies the weakness detected by the initial review. The new preliminary studies demonstrate the potential for direct and immediate benefits in ACI. Many of the reagents and technologies used in the grant are recently developed by the PI and not currently available elsewhere. The project complements the funded clinical trial of ACI at the University of Michigan (CA69102) and will enhance the scientific return from this project. Finally, the collaboration between the PI and co-PI will enhance productivity and ensure the rapid transfer of information from a pre-clinical to a clinical setting.
