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Specific targeting of glioblastoma with an oncolytic virus expressing a cetuximab-CCL5 fusion protein via innate and adaptive immunity

Nature Cancer volume 3, pages 1318–1335 (2022)Cite this article

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Abstract

Chemokines such as C-C motif ligand 5 (CCL5) regulate immune cell trafficking in the tumor microenvironment (TME) and govern tumor development, making them promising targets for cancer therapy. However, short half-lives and toxic off-target effects limit their application. Oncolytic viruses (OVs) have become attractive therapeutic agents. Here, we generate an oncolytic herpes simplex virus type 1 (oHSV) expressing a secretable single-chain variable fragment of the epidermal growth factor receptor (EGFR) antibody cetuximab linked to CCL5 by an Fc knob-into-hole strategy that produces heterodimers (OV-Cmab-CCL5). OV-Cmab-CCL5 permits continuous production of CCL5 in the TME, as it is redirected to EGFR+ glioblastoma (GBM) tumor cells. OV-Cmab-CCL5 infection of GBM significantly enhances the migration and activation of natural killer cells, macrophages and T cells; inhibits tumor EGFR signaling; reduces tumor size; and prolongs survival of GBM-bearing mice. Collectively, our data demonstrate that OV-Cmab-CCL5 offers a promising approach to improve OV therapy for solid tumors.

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Fig. 1: Construction and characterization of Cmab-CCL5 and OV-Cmab-CCL5.
Fig. 2: Cmab-hCCL5 promotes migration of human immune cells in vitro.
Fig. 3: Cmab-hCCL5 increases human NK cell-mediated ADCC against EGFR+ tumor cells.
Fig. 4: Cmab-hCCL5 enhances human macrophage ADCP against GBM cells.
Fig. 5: OV-Cmab-hCCL5 improves oncolytic virotherapy in a xenograft GBM animal model.
Fig. 6: Cmab-mCCL5 promotes murine immune cell migration and activation; OV-Cmab-mCCL5 prolongs survival of GBM-bearing mice in an immunocompetent mouse model.
Fig. 7: OV-Cmab-mCCL5 induces infiltration of innate and adaptive murine immune cells into the GBM TME.
Fig. 8: Improvement of OV-Cmab-mCCL5 correlates to immune cell activation and requires NK cells, macrophages and T cells.

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Data availability

Source data for Figs. 1–8 and Extended Data Figs. 1–10 are provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (NIH) (NS106170, AI129582, CA247550, CA264512, CA266457 and CA223400 to J.Y.; CA210087, CA265095 and CA163205 to M.A.C.), the Leukemia and Lymphoma Society (1364-19 to J.Y.) and a 2021 Exceptional Project Award from Breast Cancer Alliance (to J.Y.). The authors appreciate the Pathology Core of Shared Resources at City of Hope Beckman Research Institute and National Medical Center for performing H&E and immunohistochemical staining experiments.

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Author notes

  1. These authors contributed equally: Lei Tian, Bo Xu.

Authors and Affiliations

  1. Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Los Angeles, CA, USA

    Lei Tian, Bo Xu, Yuqing Chen, Zhenlong Li, Jing Wang, Rui Ma, Shuai Cao, Michael A. Caligiuri & Jianhua Yu

  2. Department of Computational and Quantitative Medicine, City of Hope National Medical Center, Los Angeles, CA, USA

    Jianying Zhang

  3. Department of Immunology and Theranostics, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Los Angeles, CA, USA

    Weidong Hu

  4. Department of Neurosurgery, Brigham and Women’s Hospital and Harvey Cushing Neurooncology Laboratories, Harvard Medical School, Boston, MA, USA

    E. Antonio Chiocca

  5. Georgia Cancer Center, Augusta University Medical Center, Augusta, GA, USA

    Balveen Kaur

  6. Hematologic Malignancies Research Institute, City of Hope National Medical Center, Los Angeles, CA, USA

    Michael A. Caligiuri & Jianhua Yu

  7. Department of Immuno-Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Los Angeles, CA, USA

    Jianhua Yu

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Contributions

J.Y., M.A.C. and L.T. conceived and designed the project. L.T., B.X., Y.C., Z.L., J.W., R.M., S.C. and W.H. conducted experiments. L.T. and J.Z. performed data analyses. L.T. and J.Y. wrote the paper. L.T., J.Y., M.A.C., E.A.C. and B.K. reviewed and/or revised the paper. J.Y. and M.A.C. acquired funding. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Michael A. Caligiuri or Jianhua Yu.

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Competing interests

M.A.C., J.Y., L.T., B.K. and E.A.C. have relevant or nonrelevant oncolytic virus patents awarded or pending. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Concentration of CCL5 secreted by GBM cells, and expression of CCL5 receptors on NK cells, macrophages, and T cells.

(a) Concentration of CCL5 secreted by human GBM cell lines (U251T2, Gli36ΔEGFR, and GBM30), measured by ELISA (n = 3 independent experiments). (b-e) CCR1 and CCR5 expression on human NK cells (b, n = 6 independent donors), macrophages (c, n = 5 independent donors), CD4+ T cells (d, n = 3 independent donors), and CD8+ T cells (e, n = 3 independent donors), measured by flow cytometry. Error bars indicate the standard deviations (s.d.) and data are presented as mean ± s.d..

Source data

Extended Data Fig. 2 Comparison between Cmab-hCCL5 and hCCL5-Cmab and characterization of OV-Cmab-hCCL5.

(a, b) Detection of purified Cmab-hCCL5 or hCCL5-Cmab bound to U251T2 cells, measured by flow cytometry after staining Cmab-hCCL5- or hCCL5-Cmab-incubated tumor cells with anti-Fc-APC (a) or anti-hCCL5-APC (b). Cmab-hCCL5 and hCCL5-Cmab were purified from lentivirus-infected CHO cells. IgG1 isotype served as control. (c, d) Detection of Cmab-hCCL5 in the supernatant, collected from OV-Cmab-hCCL5-infected U251T2 GBM cell culture, that was associated with U251T2 cells, measured by flow cytometry after staining supernatant-incubated U251T2 cells with anti-Fc-APC (c) or anti-hCCL5-APC (d). (e) Cmab-hCCL5 produced in the supernatant of OV-Cmab-hCCL5-infected U251T2 cells at different MOIs (n = 3 independent experiments). (f) Cell viabilities of OV-Cmab-hCCL5 infection at three indicated MOIs were measured at 24 hours post infection (hpi), 48 hpi, and 72 hpi (n = 3 independent experiments). (g) The ability of OV-Q1 and OV-Cmab-hCCL5 to induce oncolysis of GBM cells, measured by real-time cell analysis (RTCA) The experiment was performed twice with similar data. (h) U251T2 cells were infected with OV-Q1 or OV-Cmab-hCCL5 at an MOI of 2. The supernatant was harvested at the indicated time points to assess viral production using a plaque assay with Vero cells (n = 3 independent experiments). Experiments in a-d were representative of three independent experiments with similar results. Error bars indicate the standard deviations (s.d.), and data are presented as mean ± s.d. (e, f, h). Statistical analyses were performed by one-way ANOVA with P values corrected for multiple comparisons by Bonferroni method (e).

Source data

Extended Data Fig. 3 Migration of immune cells induced by OV-Cmab-hCCL5.

NK cells, macrophages, CD4+ T cells, and CD8+ T cells in the upper chamber induced by OV-Cmab-hCCL5- or OV-Q1-infected U251T2 GBM cells at an MOI of 2 in the lower chamber, measured by transwell assay. 72 hours after the transwell assay was set up, immune cells in the lower chamber were quantified by flow cytometry. Error bars indicate the standard deviations (s.d.) and data are presented as mean ± s.d.. Statistical analyses were performed by one-way ANOVA with P values corrected for multiple comparisons by Bonferroni method (n = 3 independent donors).

Source data

Extended Data Fig. 4 OV-Cmab-hCCL5 improves oncolytic virotherapy in nude mice bearing GBM30 cells.

(a) Survival of GBM30 tumor-bearing nude mice treated with OV-Q1, OV-Cmab-hCCL5, or vehicle control. Survival was estimated by the Kaplan–Meier method and compared by log-rank test (n = 7 mice). (b) Luciferase imaging of GBM30-FFL GBM mice with the indicated treatments 15 and 20 days post tumor implantation.

Source data

Extended Data Fig. 5 Construction and characterization of OV-Cmab-mCCL5.

(a, b) Detection of purified Cmab-mCCL5 bound to CT2A-hEGFR cells, measured by flow cytometry after staining Cmab-mCCL5-incubated tumor cells with anti-Fc-APC (a) or anti-mCCL5-PE (b). Cmab-mCCL5 was purified from lentivirus-infected CHO cells. IgG1 isotype served as control. (c) mCCL5 and human Fc levels in Cmab-mCCL5 in the concentrated supernatant from engineered CHO cells or OV-Q1- or OV-Cmab-mCCL5-infected CT2A-hEGFR cells, detected by immunoblotting. (d, e) mCCL5 (d) and Cmab (e) of the Cmab-mCCL5 fusion protein in the supernatant from OV-Cmab-mCCL5-infected CT2A-hEGFR cells, quantified by ELISA. Cmab-mCCL5 purified from engineered CHO cells with known concentrations served as standards (n = 3 independent experiments). (f) Migration of murine NK cells, macrophages, CD4+ T cells, and CD8+ T cells induced by Cmab-mCCL5 purified from engineered CHO cells, measured using a transwell assay. Recombinant mCCL5 (rmCCL5, 100 ng/ml) served as positive control. Experiments in a-c were repeated with three independent experiments with similar results. Error bars indicate the standard deviations (s.d.), and data are presented as mean ± s.d. (d-f). Statistical analyses were performed by 2-sided Student’s t test (f, n = 3 mice/ biologically independent samples; rmCCL5 versus control and Cmab-mCCL5 versus IgG1 isotype are only compared).

Source data

Extended Data Fig. 6 Cmab-mCCL5 promotes innate and adaptive immunocyte activation in vitro.

(a) Cytotoxicity of mouse primary NK cells against Cmab-mCCL5 pre-treated-CT2A-hEGFR cells, measured by 51Cr release. Control versus Cmab-mCCL5, P = 0.0009. A linear mixed model was used to account for the underlying variance and covariance structure (n = 3 mice). (b) CD69 expression, measured by flow cytometry, on mouse primary NK cells co-cultured with CT2A-hEGFR cells in the presence of Cmab-mCCL5 purified from engineered CHO cells or IgG1 isotype control (n = 3 independent mice). (c) ADCP of mouse macrophages induced by Cmab-mCCL5 purified from engineered CHO cells, targeting CT2A-hEGFR cells. CT2A-hEGFR target cells were prelabeled with CFSE and then were co-cultured with mouse macrophages in the presence of Cmab-mCCL5 purified from engineered CHO cells or IgG1 isotype control. The percentage of mouse macrophages that had phagocytosed labeled tumor cells was measured by flow cytometry, determined by CFSE+ macrophages (n = 3 independent mice). (d) Cytokine RNA expression levels, measured by real-time RT-PCR, of mouse macrophages co-cultured with CT2A-hEGFR cells at a ratio of 1:1 with or without Cmab-mCCL5 for 6 hours. Macrophages were purified by cell sorting to extract total RNA for generating cDNA for real-time RT-PCR (n = 3 independent mice). Error bars indicate the standard deviations (s.d.) and data are presented as mean ± s.d. (a-d). Statistical analyses were performed by one-way ANOVA with P values corrected for multiple comparisons by Bonferroni method (n = 3 mice).

Source data

Extended Data Fig. 7 OV-Cmab-mCCL5 prolongs the survival of glioblastoma-bearing mice in an immunocompetent mouse model.

(a) Survival of CT2A-hEGFR tumor-bearing mice treated with OV-Q1, OV-Cmab-mCCL5, or vehicle control. Survival was estimated by the Kaplan–Meier method and compared by log-rank test (n = 7 or 8 mice). Of note, tumors remained in the OV-Cmab-mCCL5-treated group on the day of mouse sacrifice (day 50). (b) C57BL/6J mice bearing CT2A-hEGFR cells were intracranially treated with 2 × 105 PFU of indicated oncolytic virus or vehicle control (saline) 3 days post tumor implantation. Eleven days post tumor implantation, brains were collected from mice treated with saline, OV-Q1, or OV-Cmab-mCCL5 for standard H&E staining. Data of three mice in each group are shown. Scale bar, 2 mm. (c) Scheme for main Fig. 6f. Mice bearing CT2A-hEGFR cells were treated with saline, OV-Cmab-mCCL5, OV-Q1 + mCCL5 delivered by an osmotic pump, or OV-Q1+ Cmab-mCCL5 delivered by an osmotic pump.

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Extended Data Fig. 8 Production of mCCL5 and oHSV in the GBM TME.

(a) Cmab-mCCL5 production in the brains collected from GBM mice treated with the indicated different doses of OV-Cmab-mCCL5. Mice were sacrificed on day 3 post treatment. Error bars indicate s.d., and statistical analyses were performed by one-way ANOVA with P values corrected for multiple comparisons by Bonferroni method (n = 5 mice in the saline and 2 × 105 PFU groups and n = 4 mice in the 1 × 105 PFU and 4 × 105 PFU groups). (b, c) Immunohistochemical (IHC) analysis of mCCL5, oHSV, and H&E staining of the brains collected from mice treated with saline, OV-Q1, or OV-Cmab-mCCL5. Slides with brain tissue isolated from the experimental mice were subjected to H&E and IHC staining, and anti-HSV antibody and anti-mCCL5 antibody are used for detecting mCLL5 production and oHSV (OV-Q or OV-Cmab-mCCL5) existence, respectively. Images with high and low magnification are shown in (b, scale bars, 100 μm) and (c, scale bar, 2 mm), respectively. The boxed images in (c) are shown at higher power in (b).

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Extended Data Fig. 9 OV-Cmab-mCCL5 inhibits tumor EGFR signaling and promoted the infiltration of innate and adaptive murine immune cells into the GBM TME in a dose-dependent manner.

(a) Immunoblotting of p-AKT and p-EGFR from protein harvested from the brains of mice treated with OV-Q1, OV-Cmab-mCCL5, or saline (n = 3 independent mice). (b) Total number of immune cells in main Fig. 7b-d, measured by flow cytometry (n = 4 independent mice in the OV-Q1 group and n = 5 independent mice in the OV-Cmab-mCCL5 group and the saline group). (c, d) Representative flow cytometry data of immune cells infiltrated into the brains of mice treated with saline, OV-Q1, or OV-Cmab-mCCL5. Summary data are shown in main Fig. 7c. (e) Immune cell infiltration into the brains of mice treated with indicated different doses of OV-Cmab-mCCL5 for two days (n = 5 independent mice in each group). (f) Survival of CT2A-hEGFR GBM-bearing mice treated with indicated different doses of OV-Cmab-mCCL5. Saline served as control. Survival was estimated by the Kaplan–Meier method and compared by log-rank test (n = 7 independent mice in the 2 × 105 PFU group and n = 6 in all other groups). For panels b and e, error bars indicate s.d. and data are presented as mean ± s.d., and statistical analyses were performed by one-way ANOVA with P values corrected for multiple comparisons by Bonferroni method. Experiments in c and d were representative results of one of four to five mice in each group with similar data.

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Extended Data Fig. 10 OV-Cmab-mCCL5 improves abscopal control of GBM in brain.

(a) Representative MRI images of a bilateral CT2A-hEGFR immunocompetent GBM model treated with saline, OV-Q1, or OV-Cmab-mCCL5 only on the right side of the brain. Data of two out of three to four mice in each group with similar data are shown. (b) The total number of immune cells, NK cells, macrophages, T cells, CD4+ T cells, and CD8+ T cells as well as the ratio of CD4+ T cells to CD8+ T cells in the untreated left side of the brain of the mice with the right side of the brain treated as indicated, measured by flow cytometry. Error bars indicate the standard deviations (s.d.) and data are presented as mean ± s.d.. Statistical analyses were performed by one-way ANOVA with P values corrected for multiple comparisons by the Bonferroni method (n = 5 independent mice in each group). (c) Therapeutic effects of OV-Cmab-mCCL5 when treating the two-side model with 1-day or 3-day intervals. Survival was estimated by the Kaplan–Meier method and compared by log-rank test (n = 6 mice).

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Tian, L., Xu, B., Chen, Y. et al. Specific targeting of glioblastoma with an oncolytic virus expressing a cetuximab-CCL5 fusion protein via innate and adaptive immunity. Nat Cancer 3, 1318–1335 (2022). https://doi.org/10.1038/s43018-022-00448-0

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