Immunological response and overall survival in a subset of advanced renal cell carcinoma patients from a randomized phase 2/3 study of naptumomab estafenatox plus IFN-α versus IFN-α

Abstract

Naptumomab estafenatox/ABR-217620/ANYARA (Nap) has been evaluated in clinical phase 1 and 2/3 studies. RCC patients in the phase 2/3 trial were randomized 1:1 in an open label study to receive Nap+IFN-α or IFN-α. In this study, we analyzed the UK patients for their immunological response in relation to prolonged overall survival (OS). We found that Nap-specific T cells were reduced after 3 treatment days in patients' peripheral blood. Levels of both Nap-specific CD4+ and CD8+ T cells were significantly higher 8 days after the first treatment. Patients with such pattern of reduction and expansion of Nap-binding T cells also showed increased levels of IL-2 and IFN-γ in plasma 3 hours after the first Nap treatment. In addition, Nap caused an increase of IL-6, IL-10 and TNF-α. The patients in the UK subset showed a tendency of OS benefit after Nap treatment. Most Nap treated patients with long OS had low baseline IL-6 and normal levels of anti-SEA/E-120 antibodies. Furthermore, patients with pronounced Nap induced IL-2 and T cell expansion had long OS. In conclusion, patients with low baseline IL-6 and normal anti-SEA/E-120 may respond well to Nap by T cell activation and expansion paving the way for anti-tumour effects.

Figure 1. Flow cytometric and overall plots showing the percentage of Nap-specific CD4⁺ and CD8⁺ (CD3⁺CD4⁻) T cells in PBMCs of patients pre- and several time points after start of Nap treatment

Cells were gated on lymphocytes and CD3⁺ T cells. Flow cytometric plots for patient 101–13 are shown in (A). Absolute number of Nap-specific T cells was roughly estimated using the absolute numbers obtained from full blood counts. The overall percentages and absolute numbers of Nap-specific T cells for patient 101–13 are shown in (B and C), respectively. Box and whiskers plots showing minimum, maximum, lower and upper quartiles and median of CD3⁺CD4⁺Nap⁺ T cells (D) and CD3⁺CD8⁺Nap⁺ T cells (E) for all patients before and during/following Nap treatment. Means are shown with diamonds and the forks depict p-values for differences between time points.

Figure 2. The overall percentages of Nap-specific T cells for the four patients with the most pronounced changes in these subsets are shown before and during/after the first cycle of treatment

The flow cytometric plots show the levels of Nap-specific T cells of patient 101–01 before and three time points after treatment. Prognosis and clinical responses for these patients are depicted.

Figure 3. Expression of CD45RO and CD62L within CD4⁺Nap^+/− T cells

Examples of flow cytometric plots in PBMCs of patient 101–13 pre and two time points during cycle 1 of treatment are shown in (A and D). Cells were gated on lymphocyte and CD4⁺ T cells. The figures show the mean percentage +/− SEM of CD45RO^+/− within CD4⁺Nap⁺ (B) and CD4⁺Nap⁻ T cells (C); and the mean percentage +/− SEM of CD62L^+/− within CD4⁺Nap⁺ (E) and CD4⁺Nap⁻ T cells (F) for all patients before and at all investigated time points during/following treatment.

Figure 4. Foxp3 expression within CD4⁺Nap^+/− T cells

Example of flow cytometric plots in PBMCs of patient 101–13 pre and two time points during cycle 1 of treatment is shown in (A). Cells were gated on lymphocytes and CD4⁺ T cells. The figure shows the mean percentage +/− SEM of cells having Foxp3 expression within CD4⁺Nap⁺ and CD4⁺Nap⁻ T cells (B) for all patients before and at all investigated time points during/following treatment.

Figure 5. Cytokine response (pg/mL) in plasma at pre-dose and 3 hours after the first and second day of 3 cycles of Nap treatment. Green frame shows Good MSKCC risk score and red frame shows Intermediate MSKCC risk score

(A): IL-2 response in 17 of 18 patients from the UK. The patients were categorized according to their OS. Patients having over median of baseline anti-SEA/E-120 (High anti-S; > 53.5 pmol/mL) or IL-6 (High IL-6; > 7 pg/mL) are depicted. (B): IL-2, IFN-γ, TNF-α, IL-6 and IL-10 and response in the four patients with the most pronounced Nap-specific T lymphocyte reduction on day 4 and expansion on day 8 (patients 101–01, 101–11, 101–13 and 106–01).

Figure 6. Kaplan-Meier OS plots for the UK patients

(A) All UK-patients. HR (95% CI) = 0.56 (0.26, 1.19), p = 0.13 stratified for MSKCC risk score. The four patients with the most pronounced Nap-specific T lymphocyte reduction on day 4 and expansion on day 8 (patients 101–01, 101–11, 101–13 and 106–01) are depicted. (B) Intermediate MSKCC risk score patients. HR (95% CI) = 1.03 (0.36, 2.95), p = 0.95. Filled circle shows patient having below median of baseline anti-SEA/E-120 (<53.5 pmol/mL) and IL-6 (<7 pg/mL). (C) Good MSKCC risk score patients. HR (95% CI) = 0.32 (0.11, 0.93), p = 0.029. Filled circle shows patient having below median of baseline anti-SEA/E-120 (<53.5 pmol/mL) and IL-6 (<7 pg/mL). Open circle shows that baseline anti-SEA/E-120 and IL-6 is missing.