Preclinical Evaluation of Recombinant Human IL15 Protein Fused with Albumin Binding Domain on Anti-PD-L1 Immunotherapy Efficiency and Anti-Tumor Immunity in Colon Cancer and Melanoma

Abstract

Anti-PD-L1 antibody monotherapy shows limited efficacy in a significant proportion of the patients. A common explanation for the inefficacy is a lack of anti-tumor effector cells in the tumor microenvironment (TME). Recombinant human interleukin-15 (hIL15), a potent immune stimulant, has been investigated in clinical trial with encouraging results. However, hIL15 is constrained by the short half-life of hIL15 and a relatively unfavorable pharmacokinetics profile. We developed a recombinant fusion IL15 protein composed of human IL15 (hIL15) and albumin binding domain (hIL15-ABD) and explored the therapeutic efficacy and immune regulation of hIL-15, hIL15-ABD and/or combination with anti-PD-L1 on CT26 murine colon cancer (CC) and B16-F10 murine melanoma models. We demonstrated that hIL15-ABD has significant inhibitory effect on the CT26 and B16-F10 tumor growths as compared to hIL-15. hIL-15-ABD not only showed superior half-life and pharmacokinetics data than hIL-15, but also enhance anti-tumor efficacy of antibody against PD-L1 via suppressive effect on accumulation of Tregs and MDSCs and activation of NK and CD8+T cells. Immune suppressive factors including VEGF and IDO were also decreased by combination treatment. hIL15-ABD combined with anti-PD-L1 antibody increased the activity of anti-tumor effector cells involved in both innate and adaptive immunities, decreased the TME's immunosuppressive cells, and showed greater anti-tumor effect than that of either monotherapy.

Keywords: IL15; PD-L1; colon cancer; melanoma; tumor microenvironment.

Figure 1

hIL15-ABD expression, refolding and purification. (A) SDS-PAGE (upper panel) and Western blot (lower panel) analysis of hIL15-ABD expression induced with increasing concentrations of IPTG, ranging from 0, 0.05, 0.1, 0.3, 0.5, to 1 mM in transformed E. coli, BL21(DE3) at either 30 or 37 °C. (B) SDS-PAGE analysis of fractions from transformed E. coli lysates (lane 1); supernatant of the lysates after centrifugation at 10,000 rpm for 20 min (lane 2), supernatant after washing with H₂O (lane 3); supernatant after washing with 20 mM Tris-HCl (lane 4); supernatant after washing with 50 mM Tris-HCl buffer containing 2 mM EDTA and 0.1% SDS (lane 5); supernatant after washing with 50 mM Tris-HCl, 150 mM NaCl and 2 mM EDTA (lane 6); supernatant after washing with H₂O (lane 7); supernatant after washing with H₂O (lane 8); denatured inclusion body in 8 M urea (lane 9). Lane 1 to 8 each are loaded protein equal to 75 μL and lane 9 equal to 37.5 μL of culture medium. (C) Purification of solubilized inclusion body from E. coli expressing hIL15-ABD through Ni-column. The blue curve indicates the absorption at 280 nm in mAU, whereas the green line represents the concentration of imidazole. (D) SDS-PAGE and (E) western blot analysis of elution fractions number 3–6 (lane 1–4), 10, 11, and 12 (lane 5–7), and 21 (lane 9). (F, G) The purified denatured hIL15-ABD was refolded with buffer matrix listed in Table 1 and resulted L15-ABD is examined for (F) human albumin binding and (G) stimulation of CTLL-2 proliferation. The dot line indicates the average OD490 values representing the extents of viable CTLL-2 in 96-well plates cultured with refolded hIL5-ABD.

Figure 2

In vitro characterizations and pharmacokinetics of hIL15-ABD purified in large scale. (A) Lane 1 of the SDS-PAGE indicates the purified hIL1-ABD in refolding buffer number 26 (NaCl (250 mM), GSH (1 mM) and GSSG (0.1 mM) in Tris-HCl buffer (50 mM, pH 8.5)) and lane 2 represents hIL15-ABD after being condensed following the process of refolding. (B) Refolded purified hIL-15-ABD displays similar affinity for binding to both human and murine albumin, whereas there is no measurable specific binding to albumin by hIL15. (C) hIL15 and IL15-ABD demonstrate comparable EC50 values of stimulation of STAT-5 phosphorylation in CTLL-2 cells, which are 0.10 and 0.17 nM, respectively. (D) Serum concentration-time curves of hIL15 in Balb/c mice following single intraperitoneal injection of hIL15-ABD and hIL15 at 1.5 and 1.0 μg/mouse. Data are presented as mean ± SD. N = 3 at each time point.

Figure 3

hIL15-ABD induced the accumulation of CD8⁺ T cells and NK cells, but diminished Tregs and MDSCs, resulting in colon cancer growth inhibition. (A) Animal flow chart of different treatment materials is displayed. (B) Tumor volume is recorded every 3 days and (C) tumor weight is weighed after isolation from mice on day 21. (D) Flow cytometry pattern of CD11b+/Gr-1+ MDSCs isolated from BM and SP. Percentage of CD11b⁺/Gr-1⁺ MDSCs from (E) BM and (F) SP are gated and quantified by FlowJo software. (G) Flow cytometry pattern of CD4+/CD25+/FOXP+ Tregs isolated from TDLN and SP. Percentage of CD4⁺/CD25⁺/FOXP3⁺ Tregs from (H) TDLN and (I) SP. (J) Flow cytometry pattern of CD8⁺ T cells isolated from TDLN and SP. Percentage of CD8⁺ T cells from (K) TDLN and (L) SP. (M) Flow cytometry pattern of CD3^-/CD49b⁺/CD335⁺ NK cells isolated from TDLN and SP. Percentage of CD3^-/CD49b⁺/CD335⁺ NK cells from (N) SP. (O) Mice body weight are measured 3 time per week. [BM = bone marrow, TDLN = tumor-draining lymph node and SP = spleen] (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15).

Figure 3

hIL15-ABD induced the accumulation of CD8⁺ T cells and NK cells, but diminished Tregs and MDSCs, resulting in colon cancer growth inhibition. (A) Animal flow chart of different treatment materials is displayed. (B) Tumor volume is recorded every 3 days and (C) tumor weight is weighed after isolation from mice on day 21. (D) Flow cytometry pattern of CD11b+/Gr-1+ MDSCs isolated from BM and SP. Percentage of CD11b⁺/Gr-1⁺ MDSCs from (E) BM and (F) SP are gated and quantified by FlowJo software. (G) Flow cytometry pattern of CD4+/CD25+/FOXP+ Tregs isolated from TDLN and SP. Percentage of CD4⁺/CD25⁺/FOXP3⁺ Tregs from (H) TDLN and (I) SP. (J) Flow cytometry pattern of CD8⁺ T cells isolated from TDLN and SP. Percentage of CD8⁺ T cells from (K) TDLN and (L) SP. (M) Flow cytometry pattern of CD3^-/CD49b⁺/CD335⁺ NK cells isolated from TDLN and SP. Percentage of CD3^-/CD49b⁺/CD335⁺ NK cells from (N) SP. (O) Mice body weight are measured 3 time per week. [BM = bone marrow, TDLN = tumor-draining lymph node and SP = spleen] (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15).

Figure 4

hIL15-ABD facilitated anti-tumor efficacy of anti-PD-L1 antibody via enhancing apoptosis mechanism. (A) Animal flow chart of hIL15-ABD, anti-PD-L1 and combination treatment is presented. (B,C) Colon cancer (CC) tumor growth from day 0–18 and tumor photographed on day 21 are displayed. (D,E) Melanoma tumor growth from day 0–18 and tumor photographed on day 21 are displayed. Tumor weight from (F) CC and (G) melanoma on day 18 are summarized. (H) BLI and (I) quantification results from B16-F10/luc2 bearing mice are presented. Mice body weight from (J) CC (K) melanoma model is recorded every 3 days during therapy. (M,N) IHC staining images and (L,O,P) relative proteins quantification level on CC and melanoma are presented. (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15-ABD and anti-PD-L1; scale bar = 100 μm).

Figure 4

hIL15-ABD facilitated anti-tumor efficacy of anti-PD-L1 antibody via enhancing apoptosis mechanism. (A) Animal flow chart of hIL15-ABD, anti-PD-L1 and combination treatment is presented. (B,C) Colon cancer (CC) tumor growth from day 0–18 and tumor photographed on day 21 are displayed. (D,E) Melanoma tumor growth from day 0–18 and tumor photographed on day 21 are displayed. Tumor weight from (F) CC and (G) melanoma on day 18 are summarized. (H) BLI and (I) quantification results from B16-F10/luc2 bearing mice are presented. Mice body weight from (J) CC (K) melanoma model is recorded every 3 days during therapy. (M,N) IHC staining images and (L,O,P) relative proteins quantification level on CC and melanoma are presented. (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15-ABD and anti-PD-L1; scale bar = 100 μm).

Figure 5

hIP-15-ABD offer a reinforce role of increasing anti-PD-L1 antibody induced CD8+ T cells activation. (A) Flow cytometry pattern and (B) quantification results of CD8+/IFN-γ⁺ cells from TDLN. (C) Flow cytometry pattern and (D) quantification results of CD8⁺/IFN-γ⁺ cells from SP. (E) Flow cytometry pattern and (F) quantification results of CD8⁺/IL-2⁺ cells from TDLN. (G) Flow cytometry pattern and (H) quantification results of CD8⁺/IL-2⁺ cells from SP. (I) Granzyme B and CD8 immunohistochemistry (IHC) staining images and relative proteins quantification level of (J) CC and (K) melanoma are displayed. (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15-ABD and anti-PD-L1; scale bar = 100 μm).

Figure 5

hIP-15-ABD offer a reinforce role of increasing anti-PD-L1 antibody induced CD8+ T cells activation. (A) Flow cytometry pattern and (B) quantification results of CD8+/IFN-γ⁺ cells from TDLN. (C) Flow cytometry pattern and (D) quantification results of CD8⁺/IFN-γ⁺ cells from SP. (E) Flow cytometry pattern and (F) quantification results of CD8⁺/IL-2⁺ cells from TDLN. (G) Flow cytometry pattern and (H) quantification results of CD8⁺/IL-2⁺ cells from SP. (I) Granzyme B and CD8 immunohistochemistry (IHC) staining images and relative proteins quantification level of (J) CC and (K) melanoma are displayed. (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15-ABD and anti-PD-L1; scale bar = 100 μm).

Figure 6

hIP-15-ABD combined anti-PD-L1 antibody effectively trigger the accumulation and function of NK cells. (A) Flow cytometry pattern and (B) quantification results of CD3^-/CD49b⁺, CD3^-/CD335⁺ and CD3^-/CD49b⁺/CD335⁺ NK cells from SP on CC bearing BALB/c animal model. (C) Quantification results of CD3^-/CD49b⁺, CD3^-/NK1.1⁺ and CD3^-/CD49b⁺/NK1.1⁺ NK cells from SP on melanoma bearing C57BL/6 animal model. (D,E) CD3^-/CD335⁺/IFN-γ⁺ and CD3-/NK1.1⁺/IFN-γ⁺ NK cells from SP on CC and melanoma model is displayed. (F) CD49b and IFN-γ IHC staining images and relative proteins quantification level on (G) CC and (H) melanoma are shown. Expression level of secreted (I) VEGF and (J) IL15 are shown as quantification results. (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15-ABD and anti-PD-L1; scale bar = 100 μm).

Figure 6

hIP-15-ABD combined anti-PD-L1 antibody effectively trigger the accumulation and function of NK cells. (A) Flow cytometry pattern and (B) quantification results of CD3^-/CD49b⁺, CD3^-/CD335⁺ and CD3^-/CD49b⁺/CD335⁺ NK cells from SP on CC bearing BALB/c animal model. (C) Quantification results of CD3^-/CD49b⁺, CD3^-/NK1.1⁺ and CD3^-/CD49b⁺/NK1.1⁺ NK cells from SP on melanoma bearing C57BL/6 animal model. (D,E) CD3^-/CD335⁺/IFN-γ⁺ and CD3-/NK1.1⁺/IFN-γ⁺ NK cells from SP on CC and melanoma model is displayed. (F) CD49b and IFN-γ IHC staining images and relative proteins quantification level on (G) CC and (H) melanoma are shown. Expression level of secreted (I) VEGF and (J) IL15 are shown as quantification results. (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15-ABD and anti-PD-L1; scale bar = 100 μm).

Figure 7

hIP-15-ABD combined anti-PD-L1 antibody successfully suppress the accumulation of immunosuppressive cells. (A,C) Flow cytometry pattern and (B,D) quantification results of CD3^-/CD49b⁺, CD4⁺/CD25⁺/FOXP3⁺ Tregs from TDLN and SP, respectively. (E,G) Flow cytometry pattern and (F,H) quantification results of CD11b⁺/Gr-1⁺ MDSCs from BM and SP, respectively. (I) FOXP3 and IDO IHC staining images and (J,K) relative quantification of CC and melanoma are presented. (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15-ABD and anti-PD-L1; scale bar = 100 μm).

Figure 7

hIP-15-ABD combined anti-PD-L1 antibody successfully suppress the accumulation of immunosuppressive cells. (A,C) Flow cytometry pattern and (B,D) quantification results of CD3^-/CD49b⁺, CD4⁺/CD25⁺/FOXP3⁺ Tregs from TDLN and SP, respectively. (E,G) Flow cytometry pattern and (F,H) quantification results of CD11b⁺/Gr-1⁺ MDSCs from BM and SP, respectively. (I) FOXP3 and IDO IHC staining images and (J,K) relative quantification of CC and melanoma are presented. (a¹ p < 0.05, a² p < 0.01 vs. CTRL; b¹ p < 0.05, b² p < 0.01 vs. hIL15-ABD and anti-PD-L1; scale bar = 100 μm).