Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo

Abstract

Background: 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.

Methods: 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.

Results: 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-gamma were produced, whilst production of TNF-alpha, 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-gamma was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-gamma 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.

Conclusion: 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-gamma levels.

Fig. 1

Carboxy-anhydrase-IX-specific cytolysis by scFv(G250)-gene modified T cell cultures used for adoptive immunogene therapy. The upper panels show representative experiments from each individual patient. The results are expressed as % ⁵¹Cr-release (4 h assay) at eight effector-to-target (E:T) cell ratios. Target cell were derived from the CAIX⁺ RCC cell line SKRC17-MW1 clone 4, alone (dotted line, for patient #2 and #3 only), in presence of a 30-fold excess of ‘cold’ K562 cells (closed circles), and in presence of a 30-fold excess of “cold” K562 cells and 25 μg/ml (final concentration) G250mAb (open circles). The net CAIX-specific cytolysis was defined as the level of cytolysis on CAIX⁺ RCC cells (in the presence of cold K562 targets) minus the level of cytolysis on CAIX⁺ RCC cells (in the presence of cold K562 targets and blocking G250 mAb). From these data, CAIX-specific lytic units (LU₂₀) were calculated according to Pross et al. [15]. The lower panel shows the LU₂₀-values per 10⁶ scFv(G250)⁺ T cells for the individual T-cell infusions and the mean value (horizontal line) per patient. The open circles in the lower panel indicate the positive test control results. Differences between patients were assessed by Student’s t-test (two-sided); P values ≤ 0.05 are indicated. Pat indicates patient, C indicates treatment cycle

Fig. 2

Kinetics of liver enzyme disturbances in the three patients before, during and following treatment with scFv(G250)⁺ T-cells. Treatment intervals are indicated by horizontal bars; T-ly, daily infusions of retargeted T lymphocytes; IL-2, 0.5 × 10⁶ IU rIL-2 s.c. every 12 h; Corticosteroids, i.v. solumedrol (days 8–10) and oral prednisolon (days 11–36). Treatment was stopped at day 5 after the 4th T-cell infusion in patient 1 and patient 3. Patient 2 received eight T-cell infusions as per protocol. For each patient, different linear scales were used for the y-axes in order to improve legibility of the panels

Fig. 3

Carboxy-anhydrase-IX-specific cytokine production (IFN-γ, TNF-α, Il-2, IL4, IL-5 and IL-10) by scFv(G250)-gene modified T-cells in the individual T cell infusions, expressed as the net amount [ng] of cytokine produced by 10⁶ scFv(G250)⁺ T cells during 24 h of co-cultivation with the CAIX⁺ RCC cell line SKRC1-MW1 clone 4. For each cytokine, different linear scales were used for the y-axes in order to improve legibility of the panels. See further legend to Fig. 1

Fig. 4

Kinetics of circulating scFv(G250)⁺ T-cell counts (upper panels) and scFv(G250) DNA copy numbers (lower panels) in blood samples obtained before, during and following treatment with scFv(G250)⁺ T cells in patient 1 and 2. Results of patient 3 have been published elsewhere (see Ref. [14]). In the upper panels, closed circles connected by uninterrupted lines indicate data points with proportions of scFv(G250)⁺ cells above the detection limit of 6 in 10,000 CD3⁺ T cells (see Ref. [14]). In the lower panels, the dashed lines indicate the detection limit as defined by the 95th percentile of ten blood samples of healthy donors, i.e. 1 scFv(G250) DNA copy per μl. The mean number of scFv(G250) DNA copies per scFv(G250)⁺ T cell in the T cell infusions were 2.3 for patient 1, and 4.5 and 6.8 for treatment cycles 1 and 2, respectively, of patient 2. Horizontal bars indicate treatment intervals, see further legend to Fig. 2

Fig. 5

Kinetics of scFv(G250)-mediated cytotoxicity (upper row) and CAIX-specific cytokine production [IFN-γ (2nd row) and IL-5 (3rd row)] by PBMC obtained before and after therapy. PBMC were incubated with CAIX⁺ RCC cells (open triangles), and CAIX⁺ RCC cells in the presence of G250 blocking mAb (closed triangles). Specific cytolysis is expressed as LU₂₀/10⁶ PBMC and cytokine production in pg/ml. The net scFv(G250)-mediated cytolysis (closed circles) is defined as the level of cytolysis of CAIX⁺ RCC cells minus the cytolysis of CAIX⁺ RCC cells in the presence of G250 mAb. The net scFv(G250)-mediated cytokine production (closed circles) is defined as the amount of cytokine produced in presence of CAIX⁺ RCC cells minus the amount of IFN-γ produced in presence of CAIX⁺ RCC cells and G250 mAb. See also the legends to Fig. 2

Fig. 6

Kinetics of plasma cytokine levels in blood samples of individual patients obtained before, during and following immuno-gene therapy. During the period of rIL-2 administration, blood samples were drawn just before s.c. rIL-2 injection, excepted for samples marked with asterisks (** = 2 h, and * = 4 h after rIL-2 administration); dashed line, detection limit. See further the legend to Fig. 2