Anti-ErbB-2 mAb therapy requires type I and II interferons and synergizes with anti-PD-1 or anti-CD137 mAb therapy

Abstract

Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2/ErbB-2), has become the mainstay of treatment for HER2-positive breast cancer. Nevertheless, its exact mechanism of action has not been fully elucidated. Although several studies suggest that Fc receptor-expressing immune cells are involved in trastuzumab therapy, the relative contribution of lymphocyte-mediated cellular cytotoxicity and antitumor cytokines remains unknown. We report here that anti-ErbB-2 mAb therapy is dependent on the release of type I and type II IFNs but is independent of perforin or FasL. Our study thus challenges the notion that classical antibody-dependent, lymphocyte-mediated cellular cytotoxicity is important for trastuzumab. We demonstrate that anti-ErbB-2 mAb therapy of experimental tumors derived from MMTV-ErbB-2 transgenic mice triggers MyD88-dependent signaling and primes IFN-γ-producing CD8+ T cells. Adoptive cell transfer of purified T cell subsets confirmed the essential role of IFN-γ-producing CD8+ T cells. Notably, anti-ErbB-2 mAb therapy was independent of IL-1R or IL-17Ra signaling. Finally, we investigated whether immunostimulatory approaches with antibodies against programmed death-1 (PD-1) or 41BB (CD137) could be used to capitalize on the immune-mediated effects of trastuzumab. We demonstrate that anti-PD-1 or anti-CD137 mAb can significantly improve the therapeutic activity of anti-ErbB-2 mAb in immunocompetent mice.

Fig. 1.

Anti–ErbB-2 mAb therapy requires NK, CD8α+, and CD8β+ cells. (A) H2N100 tumor cells (5 × 10⁵ cells) were injected s.c. into WT and immunodeficient BALB/c mice, and treated with 100 μg of anti–ErbB-2 mAb (clone 7.16.4) or control Ig (clone MAC4) injected i.p. on days 12, 16, 19, and 23. Immunodeficient mice consisted of mice depleted of NK cells (anti-asialo GM1; **P = 0.0097 vs. 7.16.4 + cIg), CD8α+ cells (mAb 53.6.7; *P = 0.0095 vs. 7.16.4 + cIg), or CD4+ cells (mAb GK1.5). (B) Same as A, except that mice were depleted of CD8α+ (*P = 0.0097 vs. 7.16.4 + cIg) or CD8β+ cells (mAb 53.5.8; **P = 0.0097 vs. 7.16.4 + cIg; * vs. **P = 0.0079). (C) Same as A; mice cured of H2N100 tumors were challenged on the opposite flank with 5 × 10⁵ H2N100, H2N67, or H2N113 cells injected s.c. between 42 and 56 d after primary injection (new day 0 on graph). Mice received either 100 μg control Ig (MAC4) or anti-CD8β+ mAb (53.5.8) i.p. on days −1, 0, 7, 14, and 21. (D) Same as A, except that SCID mice were used (*P = 0.0097 SCID + 7.16.4 vs. WT + 7.16.4). Data are mean ± SE of five mice per group. Statistical analyses were performed at time points indicated in figure using the Mann–Whitney test. Depleting or neutralizing mAbs were injected i.p. on days 11, 12, 19, and 26.

Fig. 2.

Immune effector mechanism of anti–ErbB-2 mAb therapy. (A) H2N100 tumor cells (5 × 10⁵ cells) were injected s.c. into WT and gene-targeted perforin-deficient BALB/c mice, and treated with 100 μg anti–ErbB-2 mAb (clone 7.16.4) or control Ig (clone MAC4) injected i.p. on days 12, 16, 19, and 23 (perforin^−/− + 7.16.4 vs. WT + 7.16.4, P = 1.0). In some groups, WT and perforin-deficient mice were also injected with neutralizing anti-FasL mAb (clone MLF1; P = 1.0 vs. cIg). (B) Same as A; mice were depleted of NK cells (#P = 0.0097 vs. 7.16.4 + cIg) or treated with neutralizing mAbs to TNF (clone TN3-19.12; P = 1.0), IFNAR1 (clone MAR1-5A3; *P = 0.0095 vs. 7.16.4 + cIg), or IFN-γ (clone H22; **P = 0.0095 vs. 7.16.4 + cIg; ***P = 0.031 vs. 7.16.4 + αIFN-γ). (C) Same as A, except that H2N100 tumors were established in BALB/c-MMTV-neu transgenic mice, and mice were injected with 7.16.4 or control Ig on days 8, 10, 12, 14, 16, 18, 20, and 22 (*P = 0.0317; **P = 0.0159; ***P = 0.0079 vs. 7.16.4 + cIg). (D) Same as A except that gene-targeted, MyD88-deficient mice were used (*P = 0.0112 vs. WT + 7.16.4). (E) Same as A; mice were treated with neutralizing mAbs against IL-1R (clone JAMA147; results of one of two representative experiments are shown). (F) Same as A; WT or gene-targeted IL-4–deficient, IL-13–deficient, or IFN-γ–deficient mice were used. WT mice were treated with neutralizing mAbs against IL-17Rα (clone M751; results of one of two representative experiments are shown). Data are mean ± SE of five mice per group. Statistical analyses were performed at the time points indicated in figure using the Mann–Whitney test. Depleting or neutralizing mAbs were injected i.p. on days 11, 12, 19, and 26.

Fig. 3.

IFN-γ–producing CD8+ T cells are required for anti–ErbB-2 mAb therapy. (A) H2N113 tumor cells (5 × 10⁵ cells) were injected s.c. into WT BALB/c mice and treated with 100 μg anti–ErbB-2 mAb (clone 7.16.4) or control Ig (clone MAC4) injected i.p. on days 12, 16, 19, and 23. Mice were injected i.p. with control Ig or anti-CD8β mAb (53.5.8) and/or anti–IFN-γ mAb (H22) or IFNAR1 mAb (MAR1-5A3). (B) Same as A, except that tumor-bearing SCID mice were treated. (C) SCID mice were injected with 5 × 10⁵ H2N113 tumor cells on day 0, then received on day 7 one of the following: (i) no transfer, (ii) 10⁶ CD4+ WT cells, (iii) 10⁶ CD8+ WT cells, (iv) 10⁶ CD4+ IFN-γ-deficient cells, (v) 10⁶ CD8+ IFN-γ-deficient cells, or (vi) 10⁶ CD4+ WT cells + 10⁶ CD8+ WT cells. Data are mean ± SE of five mice per group.

Fig. 4.

Anti–PD-1 and anti-CD137 mAbs improve therapeutic activity of anti–ErbB-2 mAb. (A) H2N113 tumor cells (5 × 10⁵ cells) were injected s.c. into BALB/c mice and treated with 50 μg anti–ErbB-2 mAb (clone 7.16.4) or control Ig and/or 100 μg anti-PD1 mAb (clone RMP1-14) injected i.p. on days 12, 16, 19, and 23 (data are mean ± SE of of one of two representative experiments with five mice per group). (B) Same as A, except that mice were treated with 50 μg anti–ErbB-2 mAb (clone 7.16.4) or control Ig and/or 100 μg of anti-CD137 mAb (clone 3H3). Data are mean ± SE of five mice per group. At day 42: 7.16.4 + cIg vs. 7.16.4 + anti–PD-1, P = 0.0097; 7.16.4 + cIg vs. 7.16.4 + anti-CD137, P = 0.0097; anti–PD-1 vs. anti–PD-1 + 7.16.4, P = 0.0097; anti-CD137 vs. anti-CD137 + 7.16.4, P = 0.0097 (using Mann–Whitney test). P = 0.0003 with Kruskal–Wallis test in all groups. (C) H2N113 tumor cells (5 × 10⁵ cells) were established in BALB/c-MMTV-neu transgenic mice and mice were injected on days 8, 10, 12, 14, 16, 18, 20, and 22 with 100 μg anti–ErbB-2 mAb (clone 7.16.4) or control Ig and/or 100 μg anti-CD137 mAb or anti–PD-1 mAb on days 8, 12, 16, and 20. Data are mean ± SE of five mice per group.