Immunotherapeutic effects of intratumoral nanoplexed poly I:C

Abstract

Poly I:C is a powerful immune adjuvant as a result of its agonist activities on TLR-3, MDA5 and RIG-I. BO-112 is a nanoplexed formulation of Poly I:C complexed with polyethylenimine that causes tumor cell apoptosis showing immunogenic cell death features and which upon intratumoral release results in more prominent tumor infiltration by T lymphocytes. Intratumoral treatment with BO-112 of subcutaneous tumors derived from MC38, 4 T1 and B16-F10 leads to remarkable local disease control dependent on type-1 interferon and gamma-interferon. Some degree of control of non-injected tumor lesions following BO-112 intratumoral treatment was found in mice bearing bilateral B16-OVA melanomas, an activity which was enhanced with co-treatment with systemic anti-CD137 and anti-PD-L1 mAbs. More abundant CD8⁺ T lymphocytes were found in B16-OVA tumor-draining lymph nodes and in the tumor microenvironment following intratumoral BO-112 treatment, with enhanced numbers of tumor antigen-specific cytotoxic T lymphocytes. Genome-wide transcriptome analyses of injected tumor lesions were consistent with a marked upregulation of the type-I interferon pathway. Inspired by these data, intratumorally delivered BO-112 is being tested in cancer patients (NCT02828098).

Keywords: BO-112; Intratumoral immunotherapy; Nanoplexed poly I:C.

Fig. 1

Local injection of BO-112 exerts antitumor effects. a. Cell viability (in terms of electric impedance) of cultured tumor cell lines was measured in xCELLigence plates over time in the presence of different concentrations of BO-112 or Poly I:C as indicated, to study effects on cell viability. b. Tumor volume follow-up of in vivo engrafted syngeneic B16F10 tumors treated intratumorally with control vehicle, Poly I:C or BO-112 as indicated in the diagram. Representative photographs of mice treated with BO-112, Poly I:C or control vehicle are included as an inset. c. Individual follow-up of tumor volume means ± SD (in graphs on the right) of MC38 and 4 T1-bearing mice treated with BO-112 or control vehicle as indicated. Experiments are representative of two similarly performed. ***P < 0.001

Fig. 2

BO-112 induces immunogenic cell death. The characterization of tumor cell death (apoptosis, necrosis, immunogenic cell death) induced by BO-112 was investigated in vitro and in vivo. a. and b. B16-OVA cells (10⁵ cells/well) were cultured alone or with BO-112 or Poly IC (0.25, 0.5 and 1 μg/ml), for 24 and 48 h. a. Apoptosis and necrosis were analyzed by flow cytometry upon staining with Annexin V and 7AAD. b. Immunogenic cell death (ICD) hallmarks were analyzed by flow cytometry studying cell surface expression of MHC-I, CD95 and Calreticulin and by measuring HMGB1 release. c. B16-OVA tumor bearing mice were intratumorally treated with BO-112 or vehicle (n = 5 per group). The diagram shows the schedule of the experiment. Graphs show that intratumoral administration of BO-112 leads to a significant increase in tumor cell apoptosis and necrosis (left) and also promotes the expression of ICD-associated markers on tumor cells. *P < 0.05, **P < 0.01***P < 0.001

Fig. 3

BO-112 intratumoral injection enhances T lymphocyte infiltrates. a. Schematic representation of the experiments to surgically harvest tumors following treatment to generate cell suspensions that were analyzed by flow cytometry. b. CD8/CD4 and CD8/Treg ratios in cell suspensions. c. Percentage of CD8⁺, CD4⁺ and CD25⁺FOXP3⁺ over total intratumoral CD45⁺ leukocytes and absolute numbers per gram of tumor tissue. d. Representative microphotographs of CD4 and CD8 immunohistochemistry analyses of sections derived from B16-OVA tumors treated as indicated. Scale bar of the main microphotograph: 100 μm. Scale bar of the inset: 60 μm. Positive cells are stained in magenta. *P < 0.05, **P < 0.01***P < 0.001

Fig. 4

Immunotherapeutic effects of combinations of intratumoral BO-112 with systemic anti-CD137 or anti-PD-L1 monoclonal antibodies. a. Schematic representation of experiments in mice bearing two B16-OVA-derived tumors engrafted on opposite flanks and intratumorally treated with BO-112 only in the right lesion and with intraperitoneal administrations of immunomodulatory monoclonal antibodies as indicated. b. Tumor volume follow-up of the injected and distant tumors in the different groups of treatment. c. Mean ± SD summary indicating statistical significance of the listed comparisons. *P < 0.05, **P < 0.01***P < 0.001

Fig. 5

BO-112 intratumoral injection induces tumor-draining lymph node enlargement and increases CD8 T cells recognizing specific antigens. a. Scheme of experimental treatment showing representative size of TDLN and their total leukocyte content in the graph comparing mice treated intratumorally with BO-112 or control vehicle. b and c.: Analysis by flow cytometry of individual TDLN cell suspensions. b. CD8 to CD4 ratios and CD8/Treg ratios. c. represents the absolute number of the indicated T-cell subsets in TDLNs. d. Class I MHC tetramer stainings to identify T cells recognizing OVA-specific epitope and TRP-2 among CD8 T cells per gram of malignant tissue in mice bearing B16-OVA tumors. e Class I MHC tetramer stainings to identify the numbers OVA- and TRP2-specific CD8+ T cells in TDLN. Absolute numbers are provided for antigen-specific CD8 T cells. *P < 0.05, **P < 0.01***P < 0.001

Fig. 6

Intratumoral BO-112 induces potent type-I IFN-related transcriptomic changes. a. Mice bearing B16-OVA tumors were treated with intratumoral BO-112 or vehicle (n = 5 per group) and total RNA was extracted as indicated to be genome-wide analyzed by gene expression microarrays. Differentially expressed transcripts were obtained by Linear Models for Microarray Data (LIMMA) analysis (b). Hierarchical clustering of differentially expressed genes between both experimental conditions. Most relevant genes for immune functions are indicated as upregulated by BO-112. c. Top canonical pathways upregulated by BO-112 treatment as defined by Ingenuity Pathway Analysis of the differentially expressed transcripts. d. Heat map representing enrichment analyses of key previously described signatures for IFNα and IFNγ stimulation, for tumor cell infiltration and activation of TILs as well as T-cell effector-related transcripts

Fig. 7

Antitumor response of intratumoral BO-112 is dependent on IFNα signaling and on Batf3-dependent Dendritic Cells. a. Tumor volume follow-up of WT and IFNARKO mice bearing B16-OVA tumors that were treated with intratumoral BO-112 or vehicle (n = 6 per group) as indicated in the diagram. Individual tumor volume and tumor volume means ± SD are shown. b. Tumor volume growth of WT or Batf3^−/− (BATF3KO) mice bearing two B16-OVA-derived tumors in which one was treated with BO-112 or vehicle (n = 6 per group) as indicated in the diagram. Tumor volume means ± SD are shown in graphs on the right. *P < 0.05, **P < 0.01***P < 0.001