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Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor - PubMed

  • ️Sat Jan 01 2011

Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor

Edmund K Moon et al. Clin Cancer Res. 2011.

Abstract

Purpose: Adoptive T-cell immunotherapy with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors to adequately exert therapeutic effects.

Experimental design: The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM; a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector, and the modified T cells were used to treat established mesothelin-expressing tumors.

Results: CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T-cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T-cell tumor infiltration by day 5 compared with mesoCAR T cells. This was associated with significantly increased antitumor activity.

Conclusions: CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve antitumor efficacy in vivo.

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Figures

Figure 1
Figure 1. Expression of mesoCAR and CCR2b expression after lentivirus (LV) transduction

Bead-activated T cells (left panel), bead-activated, LV-mesoCAR transduced T cells (middle panel), or bead-activated, LV-mesoCAR and LV-CCR2b transduced T cells (right panel) were studied using two color FACS analysis. Expression of the mesoCAR is shown on the Y axis. Expression of CCR2 is shown on the X axis.

Figure 2
Figure 2. Ability of CCL2 to induce Calcium Flux and Transwell Migration in T cells
Panels A and B

. Bead-activated T cells (Panel A) or bead-activated, LV-CCR2b transduced T cells (Panel B) were loaded with the calcium sensitive dyes (Fluo 3 AM / Fura Red AM) and studied using FACS analysis over time. Cells were first exposed to 100 ng/ml of CCL2 (thin arrow) and then to ionomycin (arrowhead) to confirm equal loading.

Panel C

. Bead-activated, LV-CCR2b transduced T cells had 45% greater migration through 0.5um polycarbonate membranes toward R10 with 100ng/ml of CCL2 than untransduced bead-activated T cells.

Panel D

. M108 tumor supernatant also induced CCR2b T cell migration in a CCL2 dependent manner. The addition of anti-CCL2 Ab blocked T cell migration. Control Ab was added to media as a negative control.

Figure 3
Figure 3. Effect of CCR2b on mesoCAR T cell killing of M108 tumor cells

In vitro killing of tumor cells by T cells was assessed by coculturing mesoCAR ± CCR2b T cells with M108 tumor cells at 20:1 ratio for 4 hours in the presence and absence of CCL2. (* = p<0.05)

Figure 4
Figure 4. T cell tumor infiltration and tumor growth after intravenous injection of activated T cells

Activated T cells were injected intravenously NSG mice with large, established M108 tumors. Groups included no T cells (ctrl), bead-activated, but untransduced T cells (untransduced), bead-activated T cells transduced with mesoCAR (mesoCAR), and bead-activated T cells transduced with both mesoCAR and CCR2b (mesoCAR + CCR2b).

Panel A and B

. 5 days after T cell injection, flank tumors (Panel A) and pooled blood (Panel B) from 3 mice in each group were subjected to FACS to determine the number of human T cells. There was a significantly increased number of T cells having infiltrated the tumor in the mesoCAR + CCR2b than the mesoCAR group. (* = p <0.02)

Panel C

. Tumor size was measured over time and revealed significantly increased anti-tumor activity after injection of mesoCAR + CCR2b T cells compared to mesoCAR T cells. * indicates statistically significant differences in mean tumor size at day 22 post T cell injection. Difference in mean flank tumor size in the mesoCAR and mesoCAR + CCR2b groups was statistically significant (p=0.003). Tumor volume was calculated using the formula volume = length × width × width / 2.

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