The therapeutic effect of anti-HER2/neu antibody depends on both innate and adaptive immunity

Abstract

Anti-HER2/neu antibody therapy is reported to mediate tumor regression by interrupting oncogenic signals and/or inducing FcR-mediated cytotoxicity. Here, we demonstrate that the mechanisms of tumor regression by this therapy also require the adaptive immune response. Activation of innate immunity and T cells, initiated by antibody treatment, was necessary. Intriguingly, the addition of chemotherapeutic drugs, although capable of enhancing the reduction of tumor burden, could abrogate antibody-initiated immunity leading to decreased resistance to rechallenge or earlier relapse. Increased influx of both innate and adaptive immune cells into the tumor microenvironment by a selected immunotherapy further enhanced subsequent antibody-induced immunity, leading to increased tumor eradication and resistance to rechallenge. This study proposes a model and strategy for anti-HER2/neu antibody-mediated tumor clearance.

Figure 1. Anti-neu antibody has limited effect in vitro but has strong effect against tumor in vivo

A) WT BALB/c mice (n=5/group) were injected s.c. with 5 × 10⁵ TUBO cells and treated with 100 μg of anti-neu (α-neu) or isotype control (Ctrl) antibody on days 14 and 21. The tumor growth was measured and compared twice a week. ***, p < 0.005 compared with isotype control group after day 23. One of five representative experiments is shown. B) TUBO cells (1 × 10⁵ cells/well) were plated in a monolayer and incubated with Erlotinib (1–4 μmol/L) or anti-neu antibody (0.3–3 μg/mL). Control groups received isotype-control antibody (Ctrl). Relative proliferation, reflected by metabolic activity, was evaluated at indicated times by MTT assay and graphed as percent of isotype control. Mean±SD; *, p < 0.05; **, p < 0.005 compared with isotype control. One of two representative experiments is shown. C) TUBO bearing BALB/c mice (n=5/group) were treated with four times with 100 μg of anti-neu antibody (α-neu) every other day and with 500 μg of Erlotinib every day for 7 days from day 18. *, p < 0.05 compared with the control group from day 33; **, p < 0.01 compared with erlotinib-treated group after day 26. One of two representative experiments is shown.

Figure 2. Anti-tumor effect of antibody depends on both FcR and adaptive immune system

A) TUBO-bearing Fcγ receptor KO and WT BALB/c mice (n=6–8/group) were treated with 100 μg of anti-neu or isotype control antibody on days 14 and 21. **, p < 0.01 compared with WT isotype control group after day 29. One of three experiments is shown. B) TUBO-bearing Wt and Rag-1^−/− mice (n=5–7/group) were treated with 100 μg of anti-neu antibody (α-neu) or isotype control (Ctrl) on days 18 and 25. *, p < 0.05; **, p < 0.005 compared to isotype control groups of each mouse strain as A. One of three experiments is shown. Increase of lymphocytes inside tumor is seen in Figure S1.

Figure 3. The therapeutic effect of anti-neu antibody treatment requires CD8⁺ cells and induces memory T cell responses

A) WT BALB/c mice (n=5–10/group) were injected s.c. with 5 × 10⁵ TUBO and treated with 100 μg of anti-neu antibody (α-neu) on days 10, 17, and 24. CD8-depleting antibody (YTS169.4.2, 200μg/mouse) was administered every 3 days, starting on day 9. *, p < 0.05; **, p < 0.005 compared to anti-neu antibody-treated WT mice. One of three experiments is shown. B) Neu Tg F1 mice (n=6/group) were injected with 3 × 10⁵ TUBO cells and treated with 100 μg of anti-neu antibody (α-neu) on days 11 and 18. CD8-dpeleting antibody (YTS169.4.2, 200μg/mouse) was administered on the same days. *, p < 0.05 compared to anti-neu antibody-treated group. One of two experiments is shown. The data from other CD8-depleting antibodies is shown in Figure S2. C) Tumor-free, antibody-treated BALB/c mice (n=14 pooled from two experiments) were re-challanged s.c. with 5 × 10⁶ TUBO cells on different site from primary tumor at least 1 month after complete rejection of primary tumors. All of mice rejected the secondary tumor. One of two experiments is shown. Anti-neu antibody therapy increases IFNγ + cells in WT (D) or Tg mice (E). D) TUBO bearing mice were treated twice with 150ug of either anti-neu (n=3) or mIgG (n=3) on days 11 and 18. Mice were sacrificed 12 days after the final treatment and splecnoytes were isolated for ELISPOT analysis as described in the materials and methods. ***p<0.0001. E. Splenocytes from neu Tg F1 mice (N=3–5) treated with anti-neu or isotype control antibody were stimulated with 3T3/KB, 3T3/NKB, or TUBO cells. The ratio of splenocytes to APC was 10:1. IFN-γ–producing cells were enumerated by ELISPOT assay. Results were expressed as number of spots per 10⁶ splenocytes. *, p< 0.05; **, p< 0.005 compared with isotype control group. One of three experiments is shown for D and E. Increase of tumor infiltrated lymphocytes was also seen in human samples after antibody treatment (see Figure S2C)

Figure 4. The therapeutic effect of anti-neu antibody depends on endogenous danger signals

A) WT and Myd88^−/− BALB/c mice (n=5–7/group) were injected s.c. with 4 × 10⁵ TUBO cells and treated with 100 μg of anti-neu (α-neu) or isotype control (Ctrl) antibody on days 21 and 28. *, p<0.01 compared to anti-neu antibody treated Myd88^−/− mice. One of two experiments is shown. B) TUBO-bearing WT BALB/c mice (n=4/group) were treated with 100 μg of anti-neu antibody (α-neu) and 100 μg of neutralizing anti-HMGB-1 antibody on days 14 and 21. **, p < 0.005, compared to anti-HMGB-1 antibody-treated group. One of four experiments is shown.

Figure 5. Administration of chemotherapeutics after antibody treatment enhances primary tumor reduction but reduces immunity induced by anti-neu antibody

WT BALB/c mice (n=5–10/group) were injected s.c. with 5 × 10⁵ TUBO cells and treated with 100 μg of anti-neu antibody (α-neu) on days 11 and 16. Select chemotherapeutic agents were injected i.p. at different time points. One of three experiments is shown. (A) 100 mg/kg of Cyclophosphamide (CTX) was injected i.p. on days 16, 23, and 33. (B) 40 mg/kg of Paclitaxel (PTX) weas injected i.p. on days 14 and 19. Treated, tumor-free mice were re-challenged with 2 × 10⁶ TUBO cells when primary tumor was not detected for at least 30 days. Percent tumor-bearing mice (C) and mean tumor volume (D) are shown. Reduced T cell proliferation was seen in Figure S3.