Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo
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We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)+ T cells is reflected by changes of selected immune parameters measured in peripheral blood.
ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)+ T lymphocytes prior to infusion, and were correlated with plasma cytokine levels.
Prior to infusion, scFv(G250)+ T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX+ RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-γ were produced, whilst production of TNF-α, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-γ was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-γ increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2.
ScFv(G250)-receptor-mediated functions of the scFv(G250)+ T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-γ levels.
KeywordsImmunogene therapy Human T lymphocytes Single chain chimeric receptor Renal cell cancer Clinical study
Adoptive transfer of autologous T lymphocytes that have been gene-transduced to express antigen-specific receptors is a potentially attractive therapy to provide tumor-specific immunity to cancer patients [1, 2, 3, 4]. We have studied safety and proof of this concept in patients with metastatic renal cell cancer (RCC) [5, 6, 7]. To this end we have constructed a single-chain antibody-type (scFv) receptor based on murine monoclonal antibody (mAb) G250 . This mAb recognizes an epitope on carboxy-anhydrase-IX (CAIX) that is over-expressed by RCC cells . Following retroviral introduction of the scFv(G250) transgene into primary human T-cells, the scFv(G250) receptor is expressed on the surface of these cells, which enables them to recognize CAIX and subsequently to exert antigen-specific effector functions in terms of cytokine production and killing of CAIX+ RCC cell lines [5, 10].
In a phase I study we have treated three patients with metastatic RCC with autologous scFv(G250)+ T cells and subcutaneous (s.c.) injections of low dose interleukin-2 (IL-2). Unexpectedly, liver toxicity was encountered, after which the study was temporarily put on hold . Examination of liver tissue from patient 1 revealed a discrete cholangitis with T-cell infiltration around the bile ducts and CAIX expression on the bile duct epithelium. The observed liver toxicity, reflected by liver enzyme disturbances, was most likely to be due to specific interactions of the scFv(G250)+ infused T cells with CAIX+ epithelial cells lining the bile ducts.
We hypothesized that the liver toxicity reflects a specific immune reaction by the infused scFv(G250)+ T cells and expected that this reaction would be reflected in changes of immune parameters measured in peripheral blood during and after therapy. Therefore, we compared functions of the scFv(G250)+ T cells prior to and post infusion—the latter using peripheral blood mononuclear cells (PBMC) taken from the patients after infusion. We tested scFv(G250)-chimeric receptor-mediated functions, such as killing of CAIX+ RCC cell lines and cytokine production upon exposure to CAIX, and determined plasma cytokine levels.
Materials and methods
The inclusion and exclusion criteria for patients participating in this study have been reported previously . The main eligibility criteria were that patients had disseminated clear cell RCC not amenable for surgery, and that the primary tumor had been removed by nephrectomy and expressed CAIX.
Autologous T lymphocytes were transduced with the scFv(G250)-chimeric receptor using retroviral transduction [11, 12]. The treatment schedule consisted of i.v. infusions of autologous scFv(G250)-transduced T-cells at escalating doses of 2 × 107 cells at day 1; 2 × 108 cells at day 2; 2 × 109 cells at days 3–5 (treatment cycle 1) and 2 × 109 cells at days 17–19 (treatment cycle 2), combined with s.c. human recombinant interleukin-2 (rIL-2, Chiron, Amsterdam), 5 × 105 IU/m2 twice daily administered at days 1–10 and days 17–26. The treatment protocol had been approved by the governmental and institutional medical ethical review boards. Written informed consent was obtained from all patients.
Collection of peripheral blood mononuclear cells and plasma
Peripheral venous blood samples were drawn prior to, during and after treatment. During therapy blood was obtained prior to the first IL-2 injection unless indicated otherwise. PBMC were isolated from sodium heparin-anti-coagulated blood by Ficoll density gradient centrifugation and were cryopreserved in liquid nitrogen. Plasma was harvested from EDTA-anti-coagulated blood by centrifugation for 10 min at 1,000×g and stored at −70°C.
Flow cytometric analysis of transduced T-cell infusions and patient blood samples
The expression of the scFv(G250) receptor on transduced T-cell cultures was studied by indirect immunofluorescence using G250 anti-idiotype mAb NuH82 [10, 13]. The absolute numbers of scFv(G250)+ T-cells in the blood of patients prior to and following infusions of transduced T-cells were assessed as described .
Assessment of scFv(G250)-chimeric receptor-mediated functions in transduced T-cell infusions and patient blood samples
Cultures of scFv(G250)-gene modified T cells to be used for adoptive immunogene therapy, as well as patient PBMC obtained prior to and after infusion of gene-modified T cells, were monitored for scFv(G250)-chimeric receptor-mediated functions, i.e. specific cytotoxic activity and cytokine production, using the CAIX+ RCC cell line SKRC17- MW1 (clone 4) [10, 11]. A scFv(G250)-chimeric receptor-expressing T-cell clone (Clone 46) was used as positive control in these assays.
Cytotoxic activity was measured in a standard 4 h 51Cr-release assay, at 2,500 target cells per well and effector cell/target cell ratios (E/T) of 80, 40, 20, 10, 5, 2.5, 1.25 and 0.63:1. The activated kill (AK)-activity of the cultured T cells and patient PBMC was blocked by adding a 30-fold excess of non-51Cr-labelled (“cold”) AK-sensitive target cells (i.e. K562) to the test. The specificity of the scFv(G250)-mediated effector functions was verified by blocking CAIX on the target cells by adding G250 mAb (at a saturating final concentration of 25 μg/ml) to the target cells 15 min before addition of the effector cells [10, 11].
For comparison of level of CAIX-specific activities between the T cell infusions of the individual patients the specific cytolytic activity and cytokine production were expressed per 106 scFv(G250)+ T cells. In addition, to correct for assay variation these activities were related to the positive test control.
Assessment of cytokine levels
The concentrations of IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 in plasma samples and culture supernatants (see above) were determined by cytokine bead arrays (BD Biosciences, San Jose, CA, USA), according to suppliers’ specifications, and expressed in pg/ml. The detection limits of the assay were 2.6 pg/ml for IL-2 and IL-4, 2.5 pg/ml for IL-5, 2.8 pg/ml for IL-10 and TNF-α and 7.1 pg/ml for IFN-γ. Cytokine production by scFv(G250)-gene modified T cells was expressed as ng cytokine produced by 106 T cells per 24 h.
Assessment of transgene copy number by quantitative real-time PCR
Genomic DNA was isolated from aliquots of the transduced T-cell infusions and from blood taken prior to, during and after therapy using the QIAamp® DNA mini kit (Qiagen, Hilden, Germany). The quantitative real-time PCR was performed as described , and data is presented as number of scFv(G250) DNA copies per μl of blood.
Liver toxicity following scFv(G250)+ T cell infusions
Characteristics of transduced T-cells immediately prior to infusion
The ex vivo generation of gene-modified T-cells was highly successful [11, 12]. The proportions of scFv(G250)+ T cells among the infused T cells were 53% for patient 1, 52% and 76 % for patient 2 (treatment cycle 1 and 2, respectively), and 63% for patient 3. Due to the reduced numbers of infused T-cells and early cessation of treatment in patients 1 and 3, the total number of infused scFv(G250)+ T-cells varied widely between patients, namely 2.13 × 109 cells for patient 1, 0.85 × 109 cells for patient 2 and 0.38 × 109 cells for patient 3 .
In vitro screening of gene-modified T-cells prior to infusion demonstrated high levels of scFv(G250)-mediated cytolysis of a CAIX+ RCC cell line (Fig. 1, upper panels). The CAIX-specific cytolytic activity of the scFv(G250)+ T-cells of patient 1 [i.e. median value 372 (range 186–433) LU20 per 106 scFv(G250)+ T cells] was significantly higher than those of patient 2 [i.e. 93 (81–116) LU20 per 106 cells] and patient 3 [i.e. 88 (51–143) LU20 per 106 cells]. This pattern was also observed when test results were corrected for between-test variation (Fig. 1, lower panel). As a result, the total scFv(G250)-mediated cytolytic capacities of the T-cell infusions varied widely between patients, i.e. 792,204 LU20 for patient 1, 78,774 LU20 for patient 2 and 33,274 LU20 for patient 3 .
Carboxy-anhydrase-IX-specific cytokine production capacity of infused scFv(G250)+ T cells
As a consequence of the widely different numbers of infused scFv(G250)+ T cells between patients, the calculated capacity of scFv(G250)-mediated IFN-γ production of the infused gene-modified T-cells ranged between 89.5 μg of IFN-γ per 24 h for patient 1, 47.2 μg for patient 2 and 14.0 μg for patient 3.
Monitoring of peripheral blood samples
The treatment episodes were associated with transient lymphopenia in all three patients, probably due to the co-administered rIL-2. Circulating T-cell numbers decreased immediately upon start of therapy to reach a nadir at approximately 50% of baseline values on day 4, to normalize again when treatment was stopped (not shown).
Circulating scFv(G250)+ T-cells and scFv(G250) DNA copies
ScFv(G250)-chimeric receptor-mediated functions in patient blood samples
Blood cytokine levels
Virtually no TNF-α was detected prior to, during and after therapy except for one observation in patient 1 (4th row of panels). Finally, virtually no IL-4 responses were seen in patients 1 and 2 (lower row of panels); patient 3 stood out by detectable IL-4 levels prior to therapy, which remained at similar levels during and after treatment.
We sought in vivo evidence for scFv(G250) receptor-mediated T-cell functions as the infusion of scFv(G250)-retargeted T-cells clearly correlated in time with the induction of liver toxicity. This toxicity is likely to be the result of the specific immune reaction of the retargeted T-cells directed against CAIX expressed by epithelial cells lining the bile ducts . Here, we show that PBMC taken from patients on therapy—during which scFv(G250)+ T cells were detectable by flow cytometry and PCR—displayed similar scFv(G250) receptor-mediated functions as did scFv(G250)+ T-cells prior to infusion, such as cytokine production after exposure to CAIX and killing of CAIX+ RCC cell lines.
We focused on fluctuations in plasma cytokine levels as markers for the in vivo activity of the scFv(G250)+ T cells. ScFv(G250)+ T lymphocytes prior to infusion produced high levels of IFN-γ and moderate levels of IL-5 upon CAIX stimulation, whereas PBMC taken from patients during therapy produced only moderate levels of IFN-γ and no IL-5 upon ex vivo exposure to CAIX. On the contrary, we observed significant plasma levels of IFN-γ and IL-5 during therapy. The fluctuations in plasma IFN-γ levels varied strongly between patients, with the highest increment in patient 1 with the most pronounced liver toxicity. We have no explanation for the aberrant kinetics of plasma IFN-γ levels in patient 2; an as yet unidentified (auto) immune activation, possibly combined with a ‘high IFN-γ genotype’  given the elevated IFN-γ level prior to treatment, may have contributed to this pattern. The plasma IFN-γ levels in patient 2 normalized during the second course of treatment, which was paralleled by relatively high CAIX-specific production capacity of the inhibitory cytokine IL-10 and an increment of plasma IL-10 levels in this patient.
To determine whether the increments in plasma IFN-γ and IL-5 levels would reflect in vivo activity of the scFv(G250)+ T cells or be related to the IL-2 administrations only, we considered the following aspects.
Adoptive transfer of T cells has often been combined with administration of exogenous rIL-2 in order to promote survival of the transferred T cells, and to support and maintain T-cell activity in vivo [17, 18, 19]. Our observation that the highest levels of scFv(G250)-mediated in vitro activity of patient PBMC, in terms of both scFv(G250)-mediated cytotoxicity and cytokine production, were detected during the period of IL-2 administration, is in line with this paradigm.
Only few studies have addressed the effects of low-dose IL-2-based treatment protocols on plasma cytokine levels. Cragun et al  performed weekly assessments of serum cytokine levels in patients receiving daily injections of 3 × 106 IU/m2/day for 6 weeks and showed a gradual increase of IL-5 from week 2 of treatment onwards, whilst levels of IFN-γ, IL-4 en GM-CSF did not change. Gene expression profiling of PBMC following 3 h of in vivo or in vitro exposure to IL-2 identified 155 genes, including IL-5 and IFN-γ, whose expression levels were increased >threefold over those in non-exposed samples [21, 22]. In addition, multiplex protein analysis of RCC patient serum showed that IFN-γ and IL-5 levels increased significantly within 3 h following administration of a single high dose of rhuIL-2 (0.72 × 106 IU/kg). However, IFN-γ levels did not increase further upon repeated IL-2 administrations, in contrast to those of other cytokines such as TNF-α, IL-5 and IL-10 . The discrepancy between the short term (i.e. within 4 h) [21, 22] and long term (i.e. 24 h)  effects of IL-2 administrations on IFN-γ and IL-5 plasma levels may be related to the different stabilities of IFN-γ and IL-5 mRNA in T-cells; their half-life is ∼2 and ∼4 h, respectively [24, 25]. Given our scheme of IL-2 administrations (i.e. once every 12 h), the relatively long half-life of IL-5 mRNA may explain the gradual increment of plasma IL-5 levels. The shorter half-life of IFN-γ mRNA would preclude such an effect on plasma IFN-γ levels.
On the basis of these combined studies, we propose that the observed increments in plasma IL-5 levels are a ‘direct’ effect of the IL-2 injections, and probably do not reflect in vivo activity of the infused scFv(G250)+ T cells. On the contrary, the observed increments in plasma IFN-γ levels cannot completely be attributed to the IL-2 injections—due to the short half-life of its mRNA—and therefore would reflect in vivo activity of scFv(G250)+ T cells. This mechanism would especially be apparent in patient 1, which received the highest number of scFv(G250)+ T cells, and developed the most severe liver toxicity.
The authors thank P. van Elzakker and B. van Krimpen for their technical assistance. This work has been supported by the Dutch Cancer Foundation (grant DDHK99-1865), the European Commission grant QLK3-1999-01262, and the Cancer Research Institute, New York, (clinical investigation grant ‘Immuno-gene therapy of metastatic RCC patients’).
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