Engager T cells: a new class of antigen-specific T cells that redirect bystander T cells

doi:10.1038/mt.2014.156

. 2015 Jan;23(1):171-8.

doi: 10.1038/mt.2014.156. Epub 2014 Aug 21.

Engager T cells: a new class of antigen-specific T cells that redirect bystander T cells

Kota Iwahori¹, Sunitha Kakarla², Mireya P Velasquez³, Feng Yu⁴, Zongzhen Yi¹, Claudia Gerken¹, Xiao-Tong Song⁵, Stephen Gottschalk⁶

Affiliations

¹ 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA.
² 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA [3] Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA.
³ 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA [3] Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.
⁴ Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA.
⁵ 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA [3] Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA.
⁶ 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA [3] Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA [4] Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA [5] Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA.

PMID: 25142939
PMCID: PMC4426792
DOI: 10.1038/mt.2014.156

Engager T cells: a new class of antigen-specific T cells that redirect bystander T cells

Kota Iwahori et al. Mol Ther. 2015 Jan.

. 2015 Jan;23(1):171-8.

doi: 10.1038/mt.2014.156. Epub 2014 Aug 21.

Authors

Kota Iwahori¹, Sunitha Kakarla², Mireya P Velasquez³, Feng Yu⁴, Zongzhen Yi¹, Claudia Gerken¹, Xiao-Tong Song⁵, Stephen Gottschalk⁶

Affiliations

¹ 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA.
² 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA [3] Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA.
³ 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA [3] Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.
⁴ Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA.
⁵ 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA [3] Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA.
⁶ 1] Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA [2] Texas Children's Cancer Center, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA [3] Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA [4] Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA [5] Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA.

PMID: 25142939
PMCID: PMC4426792
DOI: 10.1038/mt.2014.156

Abstract

Adoptive immunotherapy with antigen-specific T cells has shown promise for the treatment of malignancies. However, infused T cells are unable to redirect resident T cells, limiting potential benefit. While the infusion of bispecific T-cell engagers can redirect resident T cells to tumors, these molecules have a short half-life, and do not self amplify. To overcome these limitations, we generated T cells expressing a secretable T-cell engager specific for CD3 and EphA2, an antigen expressed on a broad range of human tumors (EphA2-ENG T cells). EphA2-ENG T cells were activated and recognized tumor cells in an antigen-dependent manner, redirected bystander T cells to tumor cells, and had potent antitumor activity in glioma and lung cancer severe combined immunodeficiency (SCID) xenograft models associated with a significant survival benefit. This new class of tumor-specific T cells, with the unique ability to redirect bystander T cells, may be a promising alternative to current immunotherapies for cancer.

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Figures

**Figure 1**
**Generation of EphA2-ENG T cells.** (a) Scheme of retroviral vector (IRES, internal ribosomal entry site; mO, mOrange). (b) Transduction efficiency was determined by FACS analysis for mO of transduced (filled) and nontransduced (NT; line) T cells. Representative FACS plot and summary of all data in shown in scatter plot format (n = 23). (c) qRT-PCR for EphA2-engager mRNA of transduced and NT T cells.

**Figure 2**
**EphA2-ENG T cells secrete cytokines, proliferate and kill target cells in an antigen-specific manner**. (a) EphA2-ENG, CD19-ENG, and NT T cells were cocultured with EphA2-positive (U373, A549, K562-EphA2) or -negative (K562) tumor cells at a ratio of 10:1. After 24 hours, interferon (IFN)-γ and interleukin (IL)-2 production was determined by enzyme-linked immunosorbent assay (n = 4). (b) EphA2-ENG, CD19-ENG, and NT T cells were cocultured with EphA2-positive (U373, A549) tumor cells at a ratio of 10:1. After 7 days, number of viable T cells were enumerated by trypan blue staining (n = 4). (c) Cytotoxicity assays were performed using EphA2-ENG, CD19-ENG, and NT T cells as effectors and EphA2-positive (U373, A549, K562-EphA2) and -negative (K562) tumor cells as targets at a E:T ratio of 10:1 (mean ± SD; n = 4).

**Figure 3**
**EphA2-ENG T cells redirect bystander T cells to tumor cells**. (a) A549 cells were cocultured with or without NT T cells and media of NT T cells or EphA2-ENG T cells. After 24 hours, IFN-γ production was determined by enzyme-linked immunosorbent assay (mean ± SD; n = 4). (b) Scheme of coculture transwell assays. (c) 1 × 10⁶ NT T cells and 2.5 × 10⁵ U373 cells were plated in the bottom well, and EphA2-ENG T cells in the insert well. The number of plated EphA2-ENG T cells ranged from 10³ to 10⁶. CD19-ENG T cells in the insert well and bottom wells without NT T cells served as controls. After 48 hours, live U373 cells were visualized by crystal violet staining. The experiment was performed three times and results of one representative experiment are shown.

**Figure 4**
**Antigen-specific activation of EphA2-ENG T cells enhances their ability to redirect bystander T cells to tumor cells**. (**a,b**) NT T cells and EphA2-ENG T cells were cultured on EphA2 (activated) or HER2 (nonactivated) protein-coated plates. Phosphate-buffered saline–treated plates served as controls. (a) IFN-γ production was determined by enzyme-linked immunosorbent assay after 24 hours (mean + SD; n = 3). (b) After 72 hours, expression of EphA2-ENG mRNA was determined by qRT-PCR (mean + SD; n = 3). (c) U373.eGFP.ffLuc or BV173.ffLuc cells were cocultured with NT T cells and increasing numbers of transwell-separated activated or non-activated EphA2-ENG T cells. After 48 hours, live tumor cells were determined by luciferase assay (n = 3; duplicate assay for each donor; activated versus nonactivated T cells: for U373: P < 0.00001; for BV173: P = 0.12).

**Figure 5**
**Model of ENG T-cell mode of action**. (a) ENG T cells (light green) express and secrete engager molecules that bind to ENG and bystander (light blue) T cells. T cells are not activated since the engager molecule is monovalent. (b) Once engager bind antigen, ENG and bystander T cells are activated (dark green and dark blue respectively). Once activated, ENG T cells increase engager molecule mRNA production.

**Figure 6**
**Antigen-dependent expansion of EphA2-ENG T cells and bystander T cells *in vivo*.** (a) A549 tumor- or nontumor-bearing mice received an intravenous (i.v.) injection of an admixture of 5 × 10⁶ eGFP.ffLuc-expressing EphA2-ENG and 5 × 10⁶ NT T cells, and one intraperitoneal (i.p.) dose of IL-2 (1,500 units). Serial bioluminescence imaging was performed to track T cells (means ± SD are shown; n = 5 per group; *P < 0.05, **P < 0.01, ***P < 0.005). (b) A549 tumor-bearing mice received an i.v. injection of an admixture of 5 × 10⁶ EphA2-ENG and eGFP.ffLuc-expressing 5 × 10⁶ NT cells or 5 × 10⁶ CD19-ENG and eGFP.ffLuc-expressing 5 × 10⁶ NT cells, and one i.p. dose of IL-2 (1,500 units). Serial bioluminescence imaging was performed to track T cells (means ± SD are shown; n = 5 per group; *P < 0.05, ***P < 0.005).

**Figure 7**
**EphA2-ENG T cells have potent antitumor activity *in vivo*.** (**a–c**) Antitumor activity of EphA2-ENG T cells in U373 glioma SCID xenograft model. Seven days after intracranial injection of 1 × 10⁵ U373.eGFP.ffLuc cells, 2 × 10⁶ EphA2-ENG (n = 8) or CD19-ENG (n = 5) T cells were injected intracranially in the same site. Untreated animals served as controls (n = 5). Tumor growth was followed by bioluminescence imaging. (a) Images of representative animals; (b) Quantitative bioluminescence imaging results for each mice (radiance = photons/sec/cm²/sr) over time. There was no tumor signal outside the brain for all groups of mice during the entire length of the experiment. The region of interest (ROI) for bioluminescence quantification was therefore restricted to the head of mice; (c) Kaplan–Meier survival curve. (**d–f**) Antitumor activity of EphA2-ENG T cells in A549 lung tumor SCID xenograft model. Seven, 14, and 21 days after intravenous (i.v.) injection of 2.5 × 10⁶ A549.eGFP.ffLuc cells mice received an i.v. dose of 1 × 10⁷ EphA2-ENG (n = 5) or CD19-ENG T cells (n = 4) and an intraperitoneal (i.p.) dose of IL-2 (1,500 units). Untreated animals served as controls (n = 5). Tumor growth was followed by bioluminescence imaging. (d) Images of representative animals; (e) Quantitative, systemic bioluminescence imaging results for each mice; (f) Kaplan–Meier survival curve.

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Comment in

In vivo secretion of anti-CD3 × anti-tumor bispecific antibodies by gene-modified cells: over a decade of T-cell engagement.
Compte M, Sanz L, Álvarez-Vallina L. Compte M, et al. Mol Ther. 2015 Apr;23(4):612-3. doi: 10.1038/mt.2015.36. Mol Ther. 2015. PMID: 25849422 Free PMC article. No abstract available.
Response to "in vivo secretion of anti-CD3 × anti-tumor bispecific antibodies by gene-modified cells: over a decade of T-cell engagement".
Iwahori K, Song XT, Gottschalk S. Iwahori K, et al. Mol Ther. 2015 Apr;23(4):613. doi: 10.1038/mt.2015.37. Mol Ther. 2015. PMID: 25849424 Free PMC article. No abstract available.

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References

1. Morgan RA, Dudley ME, Rosenberg SA. Adoptive cell therapy: genetic modification to redirect effector cell specificity. Cancer J. 2010;16:336–341. - PMC - PubMed
1. June C, Rosenberg SA, Sadelain M, Weber JS. T-cell therapy at the threshold. Nat Biotechnol. 2012;30:611–614. - PMC - PubMed
1. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365:725–733. - PMC - PubMed
1. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368:1509–1518. - PMC - PubMed
1. Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012;119:2709–2720. - PMC - PubMed

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[1] Morgan RA, Dudley ME, Rosenberg SA. Adoptive cell therapy: genetic modification to redirect effector cell specificity. Cancer J. 2010;16:336–341. - PMC - PubMed

[2] Morgan RA, Dudley ME, Rosenberg SA. Adoptive cell therapy: genetic modification to redirect effector cell specificity. Cancer J. 2010;16:336–341. - PMC - PubMed

[3] June C, Rosenberg SA, Sadelain M, Weber JS. T-cell therapy at the threshold. Nat Biotechnol. 2012;30:611–614. - PMC - PubMed

[4] June C, Rosenberg SA, Sadelain M, Weber JS. T-cell therapy at the threshold. Nat Biotechnol. 2012;30:611–614. - PMC - PubMed

[5] Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365:725–733. - PMC - PubMed

[6] Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365:725–733. - PMC - PubMed

[7] Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368:1509–1518. - PMC - PubMed

[8] Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368:1509–1518. - PMC - PubMed

[9] Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012;119:2709–2720. - PMC - PubMed

[10] Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012;119:2709–2720. - PMC - PubMed

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Engager T cells: a new class of antigen-specific T cells that redirect bystander T cells

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Engager T cells: a new class of antigen-specific T cells that redirect bystander T cells

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