Human and murine IL2 receptors differentially respond to the human-IL2 component of immunocytokines

Figures & data

Figure 1. Schematic representation of hu14.18-IL2 immunocytokines. (A) Parental immunocytokine (ICp) with original intact huIL2 bound to the H-chains of the hu14.18 mAb. (B) IC35, with huIL2 bound to the L-chains of the ch14.18 mAb. (C) IC45, with a huIL2 variant missing 5 a.a. bound to the L-chains of the ch14.18 mAb. (D) ICSK, with the IL2 superkine mutein exhibiting enhanced binding to IL2Rβ bound to the L-chains of the ch14.18 mAb. Dark gray portion represents the 98% human portion of the hu14.18 mAb that recognizes GD2. Light gray portion represents the mouse framework in the chimeric mAb. Red portion represents the 2% mouse protein remaining in the antigen-binding region.

Table 1. IL2 bioactivity in the presence of anti-IL2Rα and/or anti-IL2Rβ inhibitory mAbs.

	EC50 (IL2 ng/mL)
Cells	IL2	IL2+ anti-IL2Rα	IL2+ anti-IL2Rβ	IL2+ anti-IL2Rα+ anti-IL2Rβ
Kit225	0.71	9.43	0.98	ND
Tf-1β	9.97	9.52	ND	NT
CTLL-2	1.35	3.61	1.69	ND
SCID splenocytes	35.74	34.79	ND	NT

EC₅₀ values are the average of two–three separate experiments. ND: Not determined (proliferation curve did not reach plateau, leading to ambiguous EC₅₀ values that were not considered in the calculation). NT: Not tested (no data were collected for these conditions).

Figure 2. Effect of anti-IL2Rα and anti-IL2Rβ inhibitory mAbs on IL2 induced proliferation. (A) Tf-1β, (B) Kit225, (C) SCID splenocytes, or (D) CTLL 2 cells were incubated with inhibitory mAbs. MAbs used in (A) and (B) GL439 (anti-huIL2Rα), MikB1 (anti-huIL2Rβ) and in (C) and (D) PC61 (anti-muIL2Rα) and TM-β1 (anti-muIL2Rβ), each at 10 µg/mL for 30–60 min at 4C prior to addition of IL2. In (B) and (D), a combination of species-appropriate anti-IL2Rα and anti-IL2Rβ mAbs were also tested. Cells were cultured for 72 h at 37°C and were pulse labeled with 1 µCi of ³H thymidine for 6 h. Error bars indicate SD of triplicate samples. Data are representative of 2–10 separate experiments. Statistical differences of biological significance are represented with an asterisk *p-value = <0.001.

Figure 3. Novel ICs exhibit reduced ability to induce proliferation of cells expressing βγ-IL2Rs. Comparison of IC35 and IC45 induced proliferation on: (A) Tf-1β, (B) Kit225, (C) CTLL-2, and (D) SCID splenocytes. Cells were stimulated with increasing concentrations of IL2, ICp, IC35, or IC45 and cultured for 72 h at 37°C. The counts were determined by [³H]TdR incorporation by proliferating cells. Error bars indicate SD of triplicate samples. Data are representative of two–six separate experiments. Statistical differences of biological significance are represented with an asterisk *p-value = <0.001.

Table 2. IL2 bioactivity of IL2, ICp, IC35 and IC45.

	EC50 (IL2 ng/mL)
Cells	IL2	ICp	IC35	IC45
Kit225	0.11	0.18	0.15	0.26
Tf-1β	1.76	1.91	17.73	ND
CTLL-2	0.35	3.51	3.95	11.75
SCID splenocytes	44.10	538.43	ND	ND

EC₅₀ values are the average of three separate experiments. ND: Not determined.

Figure 4. Blocking IL2Rα allows Kit225 and CTLL-2 to respond through βγ-IL2Rs. Inhibitory antibody against the α-chain of the IL2R effects proliferation of cells expressing high affinity IL2Rs (B) and (D) but not cells expressing intermediate affinity IL2Rs (A) and (C). (A) Tf-1β, (B) Kit225, (C) CTLL-2, and (D) SCID splenocytes were incubated with inhibitory mAbs at 10 µg/mL for 30–60 min at 4C prior to addition of IL2. Cells were cultured for 72 h at 37°C and were pulse labeled with 1 µCi of ³H thymidine for 6 h. Error bars indicate SD of triplicate samples. Data are representative of 2–10 separate experiments. Statistical differences of biological significance are represented with an asterisk *p-value = <0.001.

Table 3. IC bioactivity in the presence of anti-IL2Rα.

	EC50 (IL2 ng/mL)
Cells	IL2	IL2 + anti-IL2Rα	ICp	ICp + anti-IL2Rα	IC35	IC35 + anti-IL2Rα
Kit225	0.23	2.40	0.66	6.39	0.44	14.41
Tf-1β	1.41	1.67	1.77	1.06	24.86	22.54
CTLL-2	1.30	2.45	3.46	5.37	4.34	ND
SCID splenocytes	33.87	34.16	975.27	695.33	ND	ND

EC₅₀ values are the average of three–four separate experiments. ND: Not determined.

Figure 5. The “superkine” molecules within ICSK rescue the ability to elicit proliferation through βγ-IL2Rs. Comparison of IC35 and ICSK induced proliferation on: (A) Tf-1β, (B) Kit225, (C) SCID splenocytes, and (D) CTLL-2. Cells were stimulated with increasing concentrations of IL2, ICp, ICSK, or IC35 and cultured for 72 h at 37°C. The counts were determined by [³H]TdR incorporation by proliferating cells. Error bars indicate the SD of triplicate samples. Data are representative of six–eight separate experiments. Statistical differences of biological significance are represented with an asterisk *p-value = <0.001. Bold font: These values are similar when compared to one another.

Table 4. IL2 bioactivity of IL2, ICp, IC35, and ICSK.

	EC50 (IL2 ng/mL)
Cells	IL2	ICp	IC35	ICSK
Kit225	0.31	0.42	0.39	0.47
Tf-1β	2.40	0.89	46.16	0.61
CTLL-2	1.25	5.51	6.83	6.30
SCID splenocytes	36.52	907.00	ND	614.65

EC₅₀ values are the average of four–seven separate experiments. ND: Not determined.

Figure 6. IC binding to cells expressing high or intermediate affinity IL2Rs. Flow cytometry analysis of IC binding on IL2R-expressing cells. (A) Tf-1β, (B) Kit225, (C) SCID splenocytes, and (D) CTLL-2. Cells (0.2 × 10⁶ cells/sample) were incubated with increasing amounts of the different ICs (ICp, IC35, or ICSK) in 0.2 mL of staining buffer for 30–60 min at 4°C. SCID splenocytes were pre-incubated with Mouse Fc block to minimize Fc receptor mediated binding of the ICs. For detection an Alexa fluor 647-conjugated goat anti-huIgG secondary mAb was used. MFI fold increase = (MFI of IC-stained sample/MFI of secondary mAb alone). Data represent the mean ± SD from three to five separate experiments. Statistical differences of significance are represented with an asterisk *p-value = <0.05 and **p-value = <0.01.

Figure 7. ICSK leads to increased IFNγ secretion on SCID splenocytes when compared to ICp. Comparison of IL2, ICp and ICSK induced secretion of interferon-γ on SCID splenocytes. Freshly isolated SCID splenocytes were stimulated with 7 μg/mL (equivalent to 1.3 × 10³ ng/mL IL2) ICp, IC35, or ICSK for 72 h at 37°C. Supernatants were harvested and secreted IFNγ was measured via flow cytometry using a colorimetric bead array. Error bars indicate SD of five samples. Data are representative of three separate experiments. Statistical differences of biological significance are represented with an asterisk *p-value = <0.05.

Figure 8. IC35 exhibits reduced phosphorylation of STAT5 via intermediate affinity IL2Rs. Comparison of IL2, ICp, IC35, and ICSK induced phosphorylation of STAT5 on: (A) Tf-1β and (B) Kit225. Cells were stimulated with increasing concentrations of IL2, ICp, ICSK, or IC35 and incubated for 15 min at 37°C. pSTAT5 levels were determined by intracellular flow cytometry staining using anti-pSTAT5(pY694). SI = (MFI stimulated cells/MFI unstimulated cells). Error bars indicate SD of triplicate samples. Data are representative of three separate experiments. Statistical differences of biological significance are represented with an asterisk *p-value = <0.05. SI, stimulation index; MFI, mean fluorescence intensity.

Figure 9. ADCC activity is not affected by moving the IL2 molecules to the light chains of the mAb. Comparison of ICp, IC35, and ICSK-mediated ADCC on: (A) Expanded NK cells and (B) PBMCs. Effector cells were co-incubated with ⁵¹Cr-loaded M21 tumor targets in the presence of 1 μg/mL (equivalent to 195 ng/mL IL2) ICp, IC35, or ICSK for 4 h at 37°C. Supernatants were harvested and the released ⁵¹Cr from lysed target cells was measured using a gamma counter. Percentage Cytotoxicity = [(experimental release − spontaneous release)/(maximum release − spontaneous Release)]×100. Error bars indicate SD of quadruplicate samples. Data are representative of three separate experiments.

Table 5. Fold difference of the bioactivity of the ICs when compared to IL2.

	Ratio of EC50
Cells	ICp/IL2	IC35/IL2	ICSK/IL2
Kit225	1.36	1.27	1.52
Tf-1β	0.37	19.24	0.25
CTLL-2	4.42	5.48	5.06
SCID splenocytes	24.84	ND	17.52

Ratio of EC₅₀ were calculated by using the EC₅₀ values shown in Table 5. Ratio of EC₅₀ = IC EC₅₀ / IL2 EC₅₀. ND: Not determined. Bold font: These values are similar when compared to one another.