Abstract

Introduction

Adoptive T cell immunotherapy (ACT) denotes the transfer of T lymphocytes for the treatment of malignant or infectious diseases. This approach has resulted in dramatic clinical responses in different cancer types including melanoma and Epstein-Barr virus lymphomas. (1-3) Classically, ACT involved the transfer of tumor infiltrating lymphocytes (TILs) that were expanded ex vivo from tumor biopsies. However, only 30-40% of tumor biopsy specimens yield satisfactory T cell populations, the expansion time required for patient therapy is long, and the approach has been primarily useful in malignant melanoma. (4) One reason for this may be that the immunosuppressive environment of many tumors induces tolerance by the deletion or functional inactivation of TIL T cell receptors (TCRs). (5-7)

A major advance in ACT has been the ability to rapidly generate large numbers of genetically redirected T cells that target specific tumor antigens using peripheral blood lymphocytes. One approach has used insertion of recombinant T cell receptors (TcRs). (8, 9) Another approach has been the creation of “chimeric antibody receptors” (CARs). (10) CARs are cell surface molecules in which the V_H and V_L regions of a monoclonal antibody are expressed as a single-chain variable fragment (scFv) and linked to the signal transduction domain of the CD3-zeta chain. More recent versions have added additional costimulatory domains, such as CD28 and 4-1BB, fused to the CD3-zeta chain. CAR-mediated ACT has several advantages over TcR-based ACT including high affinity recognition of the tumor antigen, MHC-independent activity, as well as the ability to link additional signaling modules to one antigen recognition event in order achieve optimal T cell activation. (11, 12) Our group has focused on introducing CARs using lentiviral vectors (13) and has initiated a clinical trial using this technology.

Although ACT appears to be one of the most robust forms of immunotherapy for the treatment of tumors, the strategy requires further optimization to overcome some significant hurdles. (11, 12) An overarching issue is safety, especially in light of lethal toxicities in recent attempts at applying ACT strategy clinically. (14, 15) However, there are also a number of issues related to increasing the efficacy of the infused T cells including: i) augmenting T cell trafficking to tumor, ii) increasing survival within tumor, and iii) making sure that the T cells retain anti-tumor activity within the intra-tumoral milieu of immunosuppressive cytokines and cells (e.g. T regulatory cells and myeloid derived suppressor cells). (16-21) In patients with metastatic melanoma, persistence of tumor antigen-specific T cells after adoptive transfer correlates with tumor regression. (22)

T cell trafficking involves a complex four-step interaction between circulating lymphocytes and endothelial cells that requires initial T cell attachment to and rolling on endothelium, T cell activation on the endothelial surface, secondary adhesion, and T cell extravasation. (17) All three of these latter steps involve chemokines and chemokine receptors (CCRs). T cell trafficking can be enhanced through binding of the tumor-produced chemokines to the appropriate CCRs on the activated T cells injected. Secretion of chemokines from the tumor that do not match the expression of the appropriate CCRs on the T cells will result in suboptimal trafficking. (23) Given that the adoptively transferred T cells are being genetically modified by insertion of optimized T cell receptors or CARs, a reasonable hypothesis is that additional modifications to change chemokine receptor expression could be advantageous.

Our group is developing ACT employing T cells transduced with lentiviral vectors encoding a chimeric antibody receptor recognizing the protein mesothelin (mesoCAR T cells). Mesothelin is a surface protein that is expressed at low levels on serosal cells (i.e. on the pleura, pericardium, peritoneum, tunica vaginalis), but is highly expressed on a number of malignancies, including malignant pleural mesothelioma (MPM), ovarian cancer and pancreatic cancer (24, 25). Due to the restricted low basal levels of expression on non-malignant tissue and the ubiquitous over-expression on various tumor types, mesothelin appears to be an attractive tumor antigen target. Clinical trials are underway using unmodified or exotoxin-conjugated anti-mesothelin antibodies and have demonstrated safety and some efficacy. (26-28) We have recently demonstrated anti-tumor activity of adoptively transferred mesoCAR T cells in preclinical mouse models of MPM (29, 30) and have initiated a pilot and feasibility clinical trial using mesoCAR T cells for patients with MPM. The hypothesis for this study is that optimization of this approach requires the CCR repertoire on the injected activated mesoCAR T cells to be “matched” to the chemokines most abundantly secreted by MPM tumors. Accordingly, we determined: 1) which chemokines are consistently secreted at high levels by MPM tumor cells, 2) which CCRs are expressed in T cells activated using clinically compliant conditions, 3) if introduction of an appropriate “matching” CCR into T cells augments trafficking to tumor, 4) and if enhanced trafficking translates into improved anti-tumor efficacy. Given the clinical prospects of treating MPM patients with extensive disease, our priority was to address these issues using large, established tumors with only a single dose of CAR T cells, and without adjunct cytokine/cellular therapy.

Methods

In vivo testing of T cell trafficking and anti-tumor activity

Five million M108 cells in a solution of X-Vivo 15 media (Lonza, Basel, Switzerland) and 50% Matrigel (BD Biosciences) were subcutaneously injected in the flanks of NSG mice. After large tumors (200-300mm³) were established (usually after 4 weeks), the mice were grouped and received one of three treatments via tail-vein: 1) 20 million untransduced bead-activated T cells, 2) 20 million bead-activated T cells transduced with mesoCAR, or 3) 20 million bead- activated T cells transduced with mesoCAR and CCR2b (mesoCAR + CCR2b) (Refer to Fig. 1 for transduction efficiencies.) Tumors were harvested five days after T cell injection and digested in a solution of 1:2 DNAse:Collagenase with rotation at 37°C. Digested tumor was then filtered through 70um nylon mesh cell strainers and washed twice with PBS+1%FBS with red blood cell lysis performed with Pharm Lyse (BD Biosciences), if needed. Whole blood was also obtained by retroorbital bleeding into heparinized tubes and subjected to red blood cell lysis. One million cells were placed in standard FACS tubes and were stained for human CD45 or CD3 expression.

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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.

Results

Functional Activity of Transduced CCR2b in activated T cells

Since CCR engagement by ligand induces an increase in intracellular calcium (41), we loaded transduced and untransduced T cells with the calcium-sensitive dyes, Fura Red and Fluo-3. Calcium flux was then monitored in real-time using FACS. As shown in Figure 2A, addition of 100ng/ml CCL2 (arrow) to untransduced T cells did not induce a calcium influx, whereas the subsequent addition of the calcum ionophore, ionomycin, (arrowhead), resulted in a large increase in the fluorescence ratio indicating calcium influx. In contrast, as shown in Figure 2B, addition of 100ng/ml CCL2 (arrow) to CCR2b T cells resulted in a clear calcium spike (arrow), as did the subsequent addition of the calcium ionophore (arrowhead), demonstrating that the introduced CCR2b exhibited physiological signal transduction in T cells.

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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.

The chemotactic function of the transduced CCR2b was also tested. Figure 2C shows the results from transwell studies in which transduced or untransduced T cells were allowed to migrate through a 0.5um polycarbonate membrane towards media alone or media containing 100ng/ml CCL2 in the lower well. Whereas CCL2 only increased the migration rate of mesoCAR T cells by 0.7 ± 2.7% compared to media alone, CCL2 induced a 45 ± 4.4% increase in migration of mesoCAR + CCR2b T cells compared to media alone (p<0.001), indicating a robust functional enhancement of the CCR2b modified T cells.

The assay was repeated to evaluate migration of CCR2b T cells towards M108-conditioned supernatant (Fig. 2D). Relative to transwell migration of CCR2b T cells towards R10, supernatant from M108 induced over a 100% increase in transwell migration. Anti-CCL2 antibody caused complete inhibition of supernatant-induced migration, confirming the effect as being CCL2 dependent.

Effect of CCR2b transduction on T cell in vitro killing of M108

We next explored the susceptibility of M108 to mesoCAR and mesoCAR + CCR2b T cell-mediated lysis using in vitro killing assays. As shown in Figure 3, when we added mesoCAR T cells to luciferase-labeled tumor cells at a constant ratio of 20 lymphocytes to one tumor cell and determined the number of live tumor cells after 4 hours, the T cells were able to kill 35.5 ± 1.9% of M108 cells. Interestingly, mesoCAR + CCR2b T cells were able to kill ~20% more M108 cells than mesoCAR T cells (55.2 ± 1.6% vs. 35.5 ± 1.9%, p<0.01). Additionally, this effect was further augmented by ~13% when mesoCAR + CCR2b T cells were exposed to 100ng/ml of CCL2 during coculture (55.2 ± 1.6% vs. 68.3 ± 3.2%, p=0.02).

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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)

Effect of CCR2b transduction on T cell infiltration and anti-tumor activity in NSG mice with M108 flank tumors

We next tested the ability of each type of activated T cell to traffic into established tumors and to exert anti-tumor activity. M108 cells were injected in the flanks of four groups (10 mice per group) of NSG mice and tumors were allowed to grow to between 200 and 300 mm³. Since this typically takes about 4 weeks, the tumors were well established, and previous work has shown that the tumors are highly vascularized by this point. (29) At this time, mice were injected with either: 1) saline, 2) 20 million untransduced, bead-activated T cells, 3) 20 million bead-activated T cells transduced with mesoCAR alone, or 4) 20 million bead-activated T cells transduced with both mesoCAR and CCR2b.

Five days after tail vein injection of T cells, three tumors in each group were harvested, digested, and subjected to FACS analysis (Fig. 4A). Because prior experience revealed that relative to the number of tumor cells, very few T cells traffic to tumor at this early time point, we chose to perform FACS analysis of digested tumor instead of in vivo imaging. The abundance of human T cells present in the tumors injected with untransduced T cells (3,400 per million total cells) or with the mesoCAR T cells (4,100 per million total cells) was very low (~0.3 - 0.4%) and not significantly different. In contrast, there was a large (>12.5 fold) and significant increase in the number of infiltrated T cells in the mesoCAR + CCR2b group (51,700 per million total cells= ~5.2%) compared to the mesoCAR group (p=0.02) and/or the untransduced T cell group (p=0.02). Blood acquired via retroorbital bleeding from the three sacrificed animals was pooled and also subjected to FACS analysis (Fig. 4B). Mice in the untransduced group had 114,100 circulating T cells per million total cells and mice in the mesoCAR T cell group had 103,600 circulating T cells per million total cells. However, mice in the mesoCAR + CCR2b T cell group had only 36,500 circulating T cells per million total cells. We interpret this to indicate that the increased TILs seen in the mesoCAR + CCR2b group tumors were due to enhanced trafficking and not enhanced systemic proliferation.

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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.

There were also significant differences in tumor growth over time (Fig. 4C). The tumors in the control and untransduced T cell groups grew steadily at similar rates, reaching a size of approximately 500 mm³ by Day 22 after T cell injection. In contrast, tumor size was significantly smaller (p< 0.01) in both the mesoCAR and mesoCAR + CCR2b groups than in the control or untransduced T cell groups. Importantly, however, tumor size was significantly smaller in the mesoCAR + CCR2b compared to the mesoCAR alone group (p<0.01). In fact, at Day 22 post T cell transfer, virtually all of the tumors in mice injected with the single dose of mesoCAR + CCR2b T cells had completely regressed.

Thus, the presence of CCR2b in the mesoCAR T cells significantly increased the number of intratumoral T cells and significantly enhanced anti-tumor efficacy.

Discussion

This study showed that it was possible to enhance trafficking of adoptively transferred T cells into large, well-established, mesothelin-expressing tumors by matching the chemokine receptors expressed on the activated T-cells with the chemokines secreted by the tumor. This was accompanied by significantly enhanced anti-tumor efficacy, even in large, established tumors.

There are a number of reasons to believe that improving T cell trafficking will be advantageous. The number of injected T cells that actually enter a tumor without targeting is extremely small (in our case ~0.3% of the tumor cells). After transduction with CCR2b, T cell infiltration increased more than 12.5-fold, to represent ~5% of the tumor. Since adoptively transferred T cells must function within tumors (either through direct killing or activation of local immune cells), it seems desirable to have the largest possible number of T cells within the tumor. The rapid localization of T cells to tissues where their targeted antigen is highly expressed may also increase their ability to proliferate and thereby increase the effector to target ratio. Others have shown that early involvement of tumor-reactive T cells is critical to effective ACT. (42)

There may also be a number of important safety advantages to tumor targeting. Focusing T cell activity to tumors could allow lower doses to be administered, thus increasing the therapeutic index, since the CAR cells will traffic to tumor instead of being distributed in the periphery where they could have deleterious “on-target/off-tumor” effects. This can be seen in Figure 4, where CCR2b T cells were found in much lower number in the blood than the T cells lacking CCR2b. Minimizing “on-target/off-tumor” effects remains an important goal in light of case reports of fatalities resulting from CAR T cell treatment toxicity. (14, 15) In the example of mesothelin-targeted T cells, normal pericardial and peritoneal tissues express mesothelin, albeit at lower levels than tumor, and could be potential sources of toxicity. Finally, it is possible that improving T cell trafficking to tumors, where substantial antigen-induced local proliferation could occur, could lessen, if not eliminate, the need for the aggressive lymphodepletion regimens that are now being employed. (43, 44) Lymphodepletion using chemotherapy (e.g. cyclophosphamide, fludarabine) and whole body irradiation presents substantial side-effect risks and markedly increases the costs of current trials. Of course, this hypothesis remains speculative and will need to be studied in a murine system in which animals have an intact immune system and effects of lymphodepletion can be studied.

Our data show that T cells activated by engagement of their T cell receptors using anti-CD3/anti-CD28 beads (the method we plan to use in future clinical trials) upregulate the expression of CCR7, CCR5 (data not shown), and most strongly, CXCR3 (from 15% to 49% of cells). Similar changes in CCRs after activation have been reported in the literature. (45-47) However, the type of T cells used (i.e. TILs versus PBMCs) and the type of stimulation used (i.e. use of IL-2 versus TCR engagement) does seem to make a difference in the expression pattern observed. (23, 48, 49) For example, Brown et al. (23), reported that activation of PBMC's propagated with OKT3 antibody, irradiated allogeneic PBMC, EBV-transformed lymphoblastoid feeder cells, and 25 U/ml IL-2 resulted in T cells with high levels of CCR2 (see their Figure 4C), a result very different than ours.

The expression pattern of CCRs using costimulated T cells indicates that these T cells would mostly likely be attracted to tumors expressing the chemokines CCL5 (binding to CCR5) or CXCL9, CXCL10, or CXCL11 (binding to CXCR3). However, when we examined a large panel of mesothelioma cell lines and pleural fluids from patients with malignant mesothelioma, we found that there was very little secretion of these chemokines (data not shown). Instead we observed substantial amounts of CCL2 expressed by most (but not all) cell lines and patient specimens. The other chemokine produced by many of the cell lines was CXCL8 (interleukin 8) (data not shown). As the receptors for CXCL8 (CXCR1 and CXCR2) are not expressed on our T cells, we are also pursuing similar studies to introduce CXCR1 into T cells.

By introducing the properly matched CCR (i.e. CCR2b) into the T cells using a lentiviral vector, we were able to achieve dual expression of the CAR and the CCR in almost one quarter of the cells (Fig. 1). This is similar to the results published by Di Stasi et al. (50) using retroviral vectors for a CD30 CAR and the CXCR4 receptor. This relatively low percentage of doubly positive cells represents a technical limitation presented by the need for two independent lentiviral transductions, each with less than 100% efficiency. We did not purify these cells after activation, yet still saw impressive responses. We are now modifying our approach to use a biscistronic vector expressing both transgenes and are achieving higher dual expression levels and expect to further augment pre-clinical efficacy.

The concept of matching tumor-secreted chemokines with the appropriate CCR on the transfused, activated T cell, was first proposed by Kershaw et al. in 2002 (48) who conducted in vitro studies matching secretion of the chemokine Groα (CXCL1) by melanoma cells with a retrovirally-transduced CXCR2 receptor in IL-2-activated T cells. Augmented migration of transduced T cells toward tumor supernatant was shown in a transwell assay, but no animal experiments were reported. The importance of chemokine/CCR matching was also shown by Brown et al. (23) in experiments demonstrating that their activated T cells expressed CCR2 (unlike ours) and that these cells trafficked better to tumors from lymphoma, neuroblastoma, and melanoma cell lines that expressed high levels of CCL2. Subsequently, at least three groups have shown augmented trafficking and anti-tumor efficacy of TILs or CARs in which chemokine receptor modification has been accomplished in models of Hodgkins lymphoma (using a human CD30-CAR expressing CCR4) (50), neuroblastoma (using a human GD2-CAR expressing CCR2b) (47), and melanoma (using a mouse transgenic T cell line expressing CXCR2). (51) In these studies, expression and function of the transduced CCRs were confirmed in vitro, followed by demonstration of increased in vivo migration and a resultant slowing in tumor growth. However, in each of these studies, the modified T cells were introduced only one week or less from the time of xenograft tumor cell injection (thus before true “establishment” of the tumors), IL-2 was injected during the period of ACT, and multiple injections of T cells were given. Although IL-2 supplementation may promote survival of introduced T cells, there is now clear data showing that IL-2 also stimulates the expansion of Treg cells (52, 53) – clearly, an undesired effect, especially in MPM (54, 55). Additionally, Peng et al. (51) also injected peptide-pulsed dendritic cells and used flow cytometry-assisted sorting for CAR positive T cells, whereas we did not. We feel that our study significantly extends these previous reports by: 1) being the first report to show an enhancement of actual tumor regression (rather than just slowing of tumor growth), 2) direct enumeration of the efficiency of CAR T cell trafficking rather than using estimates from bio-imaging, 3) demonstrating that increased adoptive T cell migration and anti-tumor activity are possible in a model of large, established tumors, 4) showing clear efficacy with only one dose of genetically modified T cells, and 5) demonstrating that targeted 2^nd generation CARs can be highly effective after intravenous injection without the need for additional adjuvant cytokine or cellular therapy. Interestingly, the in vitro data from this paper suggests that CCL2/CCR2 interactions may actually augment T cell effector function independent of its effects on trafficking, and studies to uncover this mechanism are planned.

One of the goals of this study was to provide relevant preclinical data to support optimization of ongoing clinical trials. Our first target population for the mesoCAR T cells will be patients with malignant pleural mesothelioma (MPM). This disease is highly associated with previous asbestos exposure and usually presents in the fifth to seventh decade of life with dyspnea, a pleural effusion, and non-pleuritic chest pain. Current therapies are inadequate, stimulating our group and others to pursue novel immune and immune-gene therapy approaches. (25, 36, 56-59) As mentioned above, most MPM's express high levels of mesothelin (as do ovarian and pancreatic cancers). Once we have collected our baseline safety and efficacy clinical trial data, the findings from this study suggest that we can improve the approach employing tumor-targeting strategies using CCR2b or potentially other CCRs. In addition, our data suggests that the efficacy of ACT, in general, might be improved in clinical trials by “personalizing” the injected T cell chemokine receptor profile with the chemokines produced by the subject's own tumor.

Supplementary Material

Acknowledgements

REFERENCES