. 2015 Apr 13;27(4):489-501.

doi: 10.1016/j.ccell.2015.03.004.

Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2

Eric F Zhu¹, Shuning A Gai¹, Cary F Opel¹, Byron H Kwan², Rishi Surana³, Martin C Mihm⁴, Monique J Kauke¹, Kelly D Moynihan², Alessandro Angelini⁵, Robert T Williams⁶, Matthias T Stephan⁷, Jacob S Kim⁵, Michael B Yaffe⁸, Darrell J Irvine⁹, Louis M Weiner³, Glenn Dranoff¹⁰, K Dane Wittrup¹¹

Affiliations

¹ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
³ Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
⁴ Department of Pathology, Harvard Medical School, Boston, MA 02115, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA 02214, USA.
⁵ Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁶ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁷ Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁸ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Boston, MA 02129, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
¹⁰ Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
¹¹ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: wittrup@mit.edu.

PMID: 25873172
PMCID: PMC4398916
DOI: 10.1016/j.ccell.2015.03.004

Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2

Eric F Zhu et al. Cancer Cell. 2015.

. 2015 Apr 13;27(4):489-501.

doi: 10.1016/j.ccell.2015.03.004.

Authors

Affiliations

¹ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
³ Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
⁴ Department of Pathology, Harvard Medical School, Boston, MA 02115, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA 02214, USA.
⁵ Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁶ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁷ Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁸ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Boston, MA 02129, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
¹⁰ Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
¹¹ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: wittrup@mit.edu.

PMID: 25873172
PMCID: PMC4398916
DOI: 10.1016/j.ccell.2015.03.004

Abstract

Cancer immunotherapies under development have generally focused on either stimulating T cell immunity or driving antibody-directed effector functions of the innate immune system such as antibody-dependent cell-mediated cytotoxicity (ADCC). We find that a combination of an anti-tumor antigen antibody and an untargeted IL-2 fusion protein with delayed systemic clearance induces significant tumor control in aggressive isogenic tumor models via a concerted innate and adaptive response involving neutrophils, NK cells, macrophages, and CD8(+) T cells. This combination therapy induces an intratumoral "cytokine storm" and extensive lymphocyte infiltration. Adoptive transfer of anti-tumor T cells together with this combination therapy leads to robust cures of established tumors and development of immunological memory.

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Figures

**Figure 1. Extended serum half-life IL-2 combined with a tumor-specific antibody controls tumor growth**
(A–D) Tumor growth and survival curves of C57BL/6 mice bearing subcutaneous B16F10 tumors treated with (A) PBS, murine IL-2 and/or TA99; (B) PBS, Proleukin and/or TA99; (C) PBS, Fc/IL-2 and/or TA99; (D) PBS, Fc/IL-2+TA99, or MSA/IL-2+TA99. Arrows indicate points of treatment. n = 5 per group. *p < 0.05, **p < 0.01 v. corresponding color group in legend. Data are mean ± SEM. See also Figure S1 and Table S1.

**Figure 2. Extended serum half-life IL-2 combined with a tumor-specific antibody is generalizable**
Tumor growth and survival curves of (A) hmHER2 Tg mice bearing subcutaneous D5-HER2 tumors treated with PBS, Fc/IL-2 and/or Herceptin. n = 10 per group. (B) C57BL/6 mice bearing subcutaneous RM9 tumors treated with PBS, Fc/IL-2 and/or 3F8 mAb. n = 5 per group. (C) C3H mice bearing subcutaneous Ag104A tumors treated with PBS, Fc/IL-2 and/or 237 mAb. n = 5 per group. *p < 0.05, **p < 0.01, ****p < 0.0001 v. corresponding color group in legend. Data are mean ± SEM.

**Figure 3. Fc/IL-2+TA99 induces immune infiltrates within B16F10 tumors**
(A) Representative immunofluorescence of immune cell infiltrates into B16F10 treated with PBS or Fc/IL-2+TA99. Hoescht 33258 (Blue), indicated lineage marker (Red). Magnification 20×. Scale bar 100 microns. (B) Analysis of immune cell infiltrates into treated B16F10 tumors, normalized to total tumor mass, with lineage markers as indicated. n = 10 per group. *p < 0.05, **p < 0.01 between indicated groups. (C) Tumor growth and survival curves of C57BL/6 mice bearing subcutaneous B16F10 tumors treated with PBS, Fc/IL-2+TA99, and with depletions as indicated. n = 10 per group. *p < 0.05, **p < 0.01 v. corresponding color group in legend. Data are mean ± SEM. See also Figure S2.

**Figure 4. Fc/IL-2+TA99 induces intratumoral cytokine storm and uniquely increases several inflammatory and neutrophil-related factors**
(A–B) Luminex analysis of intratumoral concentrations of a panel of cytokines and chemokines in tumors treated with (A) PBS or Fc/IL-2+TA99; (B) Fc/IL-2 or Fc/IL- 2+TA99. Blue bars indicate factors elevated only in Fc/IL-2+TA99 treated tumors. *p < 0.05, **p < 0.01 v. Fc/IL-2+TA99. (C–E) Intratumoral concentrations of (C) MIP-2, (D) G-CSF, and (E) IL-6 in tumors treated with Fc/IL-2+TA99 and with depletions as indicated. n = 10 per group. (F) *Cxcl2* expression in CD11b⁺F4/80⁺ macrophages sorted from tumors treated with the indicated conditions, normalized to beta-2 microglobulin (*B2m*) expression. n = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001 between indicated groups. Data are mean ± SEM. See also Figure S3.

**Figure 5. Neutrophil infiltration and function modulated by a suite of immunological elements; oxidative burst contributes to therapeutic efficacy**
(A) Analysis of CD11b⁺Ly-6C⁺Ly-6G⁺ neutrophil infiltration into B16F10 tumors treated with PBS, Fc/IL-2+TA99, and with depletions/neutralizations as indicated, normalized to total tumor mass. Complement depleted by cobra venom factor (CVF). n = 10 per group. *p < 0.05, **p < 0.01, ***p < 0.001 v. Fc/IL-2+TA99. (B) Tumor growth and survival curves of C57BL/6 mice bearing subcutaneous B16F10 tumors treated with PBS, Fc/IL-2+TA99, and with depletions/neutralizations as indicated. n = 5 per group. *p < 0.05, **p < 0.01 v. Fc/IL-2+TA99. (C) Analysis of degranulation and activation markers of neutrophils in mice treated with Fc/IL-2+TA99. n = 5 per group. *p < 0.05, ***p < 0.001 between indicated groups. (D–E) B16F10 tumors were treated with indicated treatments and depletions/neutralizations as applicable. On days of imaging, luminol was injected and average radiant luminescence was quantified. Representative images on day 8 are shown. In (D): a) PBS; b) Fc/IL-2; c) TA99; d) Fc/IL-2+TA99. In (E): a) PBS; b) Fc/IL-2+TA99; c) Fc/IL-2+TA99+anti-CD8α; d) Fc/IL-2+TA99+anti-IFNγ. Arrows indicate points of treatment. n = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Fc/IL-2+TA99 v. corresponding color group in legend. Scale bar 1 cm. (F) Tumor growth and survival curves of C57BL/6 mice or p47phox KO mice bearing subcutaneous B16F10 tumors treated with PBS or Fc/IL-2+TA99. n = 5 per group. *p < 0.05, **p < 0.01 v. p47phox KO: Fc/IL-2+TA99. Data are mean ± SEM. See also Figure S4.

**Figure 6. Fc/IL-2 or Fc/IL-2+TA99 have comparable effect on NK cells and CD8⁺ T-cells**
(A–C) Analysis of NK cell or CD8⁺ T-cell activation markers, as a percentage of the intratumoral NK cell or CD8⁺ T-cell population, respectively. The following conditions were investigated (A) CD3NK1.1⁺KLRG1 ⁺ cells; (B) CD3⁺CD8⁺CD25⁺ cells; (C) CD3⁺CD8⁺CD69⁺ cells. (D–G) Analysis of IFNγ-producing or degranulating NK cells or CD8⁺ T-cells, as a percentage of the intratumoral NK cell or CD8⁺ T-cell population, respectively. The following conditions were investigated (D) CD3-NK1.1⁺IFNγ⁺ cells; (E) CD3-NK1.1⁺CD107a⁺ cells; (F) CD3⁺CD8⁺IFNγ⁺ cells; (G) CD3⁺CD8⁺CD107a⁺ cells. n = 10 per group. *p < 0.05, **p < 0.01, ***p < 0.001 between indicated groups. (H) Tumor growth and survival curves of C57BL/6 mice bearing subcutaneous B16F10 tumors treated with PBS, Fc/IL-2+TA99, or Fc/IL-2+TA99 with anti-IFNγ antibody. n = 5 per group. *p < 0.05 v. Fc/IL-2+TA99. Data are mean ± SEM. See also Figure S5.

**Figure 7. Pmel-1+Fc/IL-2+TA99 induces long-term control of B16F10 growth through activation and expansion of pmel-1 cells**
(A) Tumor growth and survival curves of C57BL/6 mice bearing subcutaneous B16F10 tumors were treated with indicated combinations of Fc/IL-2, TA99, and 10⁷ CD8⁺ pmel-1 T-cells. All mice received total body irradiation 5 days after tumor inoculation. n = 3–5 per group. **p < 0.01 v. pmel-1+Fc/IL-2+TA99. (B) Representative pictures 100 days after tumor inoculation showing vitiligo after treatment. a) Non-treated, non-tumor bearing and age-matched; b) pmel-1+Fc/IL-2+TA99 treated. (C) Identical to (A), except CD8⁺ T-cells were derived from luciferase expressing pmel-1 mice. On days of imaging, D-luciferin was injected and average radiant luminescence was quantified. Representative pictures on day 14 are shown: a) pmel-1; b) pmel-1+Fc/IL-2; c) pmel-1+TA99; d) pmel-1+Fc/IL-2+TA99. Arrows indicate point of treatment. n = 5 per group. *p < 0.05, ****p < 0.0001 pmel-1 v. corresponding color group in legend; #p < 0.05, ####p < 0.0001 pmel-1+TA99 v. corresponding color group in legend. Scale bar 1 cm. Data are mean ± SEM. See also Figure S6.

**Figure 8. Mechanistic model of the immune response induced by Fc/IL-2+TA99 against B16F10**
Combination of Fc/IL-2 and anti-tumor IgG administration induces a synergistic innate and adaptive immune response. Fc/IL-2 increases the sustained exposure of NK cells and CD8⁺ T-cells to IL-2 signaling, activating them. The presence of three factors— anti-tumor antibodies, Fc/IL-2, and NK cells—induces macrophages to release the neutrophil chemoattractant, MIP-2. This chemokine recruits neutrophils which, expressing FcγR, likely engage the tumor via the opsonizing antibodies and perform ADCC. These and other PMNs have their cytotoxic function bolstered by IFNγ released by the activated NK cells and CD8⁺ T-cells. Finally, NK cells and CD8⁺ T-cells perform direct effector function against the tumor cells as well. Overall, this complex network of immune responses results in controlled B16F10 growth during the course of treatment.

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Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2

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Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2

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