Multivalent Forms of the Notch Ligand DLL-1 Enhance Antitumor T-cell Immunity in Lung Cancer and Improve Efficacy of EGFR-Targeted Therapy

Abstract

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44(+)CD62L(+)CD8(+) memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity.

Figure 1. Clustered DLL1 binds to four Notch receptors, up-regulates Notch signaling and modulates expression of hematopoietic Notch genes in vivo.

(A) Precipitation of Notch receptors from mouse thymus lysate by DLL1-Fc/anti-Fc antibody or Fc/anti-Fc antibody (control) complexes bound to protein G beads; precipitated proteins were separated by Western blot and visualized using antibodies to Notch 1, 2, 3 or 4. (B–D) mRNA expression of downstream Notch target genes Hes1 and Hey1 (B), Notch ligands (C), and Notch receptors (D) in hematopoietic organs of mice treated with clustered (clust) DLL1. Mice received 3 injections of clustered DLL1 or control clusters i.p. every 2 days. Gene expression was evaluated on the second day after the last injection by qRT-PCR. Mean ± SEM, 6–8 mice per group, *p<0.05; **p<0.01. BM – bone marrow; LN – lymph nodes.

Figure 2. Enhancement of DLL1-mediated Notch signaling promotes T cell differentiation and survival in tumor-bearing host and elicits tumor antigen-specific CTL responses in tumor-bearing mice

LLC tumor-bearing mice were treated with clustered DLL1 or control clusters i.p. every 2 days for 10 days. Gene expression or Stat phosphorylation were evaluated by qRT-PCR or intracellular protein immunofluorescence staining, respectively, in a pool of splenocytes and lymph node cells; expressed as fold increase in clustered DLL1-treated mice over control clusters group. (A) Changes in phospho-Stat proteins in gated CD4⁺ cells following intracellular flow cytometry staining; (B) Representative phospho-Stat1 versus CD4 dot plot; (C) Expression of transcription factors regulating T cell differentiation assessed by qRT-PCR. Mean ± SEM, 5 mice per group, *p<0.05; **p<0.01. (D) IFN-γ-producing cells to endogenous LLC tumor antigen MUT1 were enumerated by ELISPOT assay in a mixture of splenocytes and lymph node cells of LLC tumor-bearing mice treated with control or DLL1 clusters following re-stimulation in vitro with tumor antigenic peptide MUT1 for 48 h. Mice received clustered DLL1 or control treatment for 10 days immediately after injection of LLC cells. Mean ± SEM, 5 mice per group, *p<0.05.

Figure 3. Attenuation of tumor growth by clustered DLL1 correlates with the improved anti-tumor T cell immunity

Mice inoculated with D459 tumor cells were treated with clustered DLL1 or control clusters i.p. every 2 days from day 7 (after tumors reached 4–5 mm) to day 19. (A) D459 tumor growth. Mean ± SEM, 8 mice per group, *p<0.05; **p<0.01. (B) Surface expression of activation marker CD25 and intracellular staining for IFN-γ in cultures of splenic CD8⁺ T cell isolated from mice on day 21 following re-stimulation in vitro with p53 D459 tumor antigenic or control peptide for 60 hrs. Numbers – MFI for control (blue) and cognate (red) peptide-stimulated cells. Representative histograms of total 4. (C) Proportion of memory CD44⁺CD62L⁺CD8⁺ T cells in splenocytes of mice; (D) Representative CD44 versus CD62L flow cytometry dot plot on gated splenic CD8⁺ T cells; from a total of 5 mice. (E) Increased number of IFN-γ-producing T cells within splenic CD44⁺CD62L⁺ memory CD8⁺T cell population. (F) Decreased number of regulatory T cells in spleen of clustered DLL1 compared with control cluster-treated animals. (C, E, F) Mean ± SEM, 5 mice per group, *p<0.05.

Figure 4. Multivalent DLL1 therapy elicits tumor antigen-specific T cell memory, enhances tumor infiltration by T cells and attenuates tumor angiogenesis

D459 tumor growth (A) and weight (B) in SCID-NOD mice that received lymphocyte transfer from donor mice bearing D459 tumor and treated with clustered DLL1 or control clusters. 5×10⁶ cells of the total lymphocyte fraction from a pool of RBC-depleted splenocytes and tumor-draining LN cells were harvested from donor mice at day 21 (see Fig. 3) and transferred into SCID-NOD mice bearing palpable (3–4 mm) D459 tumors at day 5. Mean ± SEM, 5 SCID-NOD mice per group, *p<0.05; **p<0.01. (C, D) Immunostaining of tumor tissues with CD3ε (C) or CD34 (D) antibodies. D459 tumor-bearing mice were treated with DLL1 or control clusters, as in Fig. 3, and tissue sections were prepared on day 21. Representative images and the numbers of infiltrating CD3ε⁺ T cells or CD34⁺ tumor vessels are presented. Mean ± SEM, 5 mice per group; 10 fields on 2 nonadjacent sections were counted for each sample; *p<0.05.

Figure 5. Multivalent DLL1 significantly improves progression-free survival (PFS) in combination with EGFR oncogene-targeted treatment in EGFRL858R transgenic mouse model

Transgenic EGFRL858R mice with induced lung tumors were treated with erlotinib in combination with clustered DLL1 or control clusters, as shown in (A). (A) Lung tumor (white opacities) growth was evaluated by MRI and volume quantified. (Inserts) Representative MRI images at the corresponding time points. (B) PFS; recurrence was determined when tumor volume exceeded by 30% residual tumor volume after erlotinib treatment. Mean ± SEM, 8 mice per group, *p<0.05, **p<0.05.

Figure 6. Clustered DLL1 enhances Notch signaling, modulates expression of Notch genes in hematopoietic compartment and provides immunological benefit in combination with EGFR inhibition in EGFRL858R transgenic lung cancer mouse model

Lung tumors were induced and mice treated with erlotinib in combination with clustered DLL1 or control clusters, as in Fig. 5. For immunological assays and analysis of lung-infiltrating immune cells, single cell suspensions from lungs were prepared on day 50 after tumor induction. (A) mRNA expression of downstream Notch target genes in CD45⁺ cells isolated from mouse lungs. (B) Protein expression of Notch ligands and receptors in splenocytes. (C) Proportion of lung infiltrating immune cell lineages. Mean ± SEM, n=5, *p<0.05.

Figure 7. DLL1-induced Notch signaling enhances human peripheral T-cell proliferation without any pro-tumorigenic effects on cancer cells in vitro

(A) PBMCs from human donors were stimulated with beads-coupled CD3, CD28, and CD137 antibodies with or without multivalent DLL1 for 4 days. Gated CD3⁺ T cell proliferation was assessed by CFSE dilution. Representative histogram overlays as well as day 4 total cell yield are shown. Mean ± SEM, n=4, *p<0.05. (B) Expression of Notch1 and Notch3 in mouse and human cancer cell lines assessed by Western blotting. Notch1 and Notch3 bands at approx. 110 kD and 90 kD, respectively, correspond to intracellular domain; higher bands at approx. 250 kD represent full length Notch (see also note for Western blot in Materials and Methods section). (C) Cell proliferation measured by [3H]-thymidine incorporation. (D) Colony formation in soft agar evaluated after 2 weeks. Mean ± SEM, n=4. Both PBMC and cancer cells were cultured with clustered DLL1 or control clusters at 1 µg/ml of DLL1-Fc protein.