Short-Term Local Expression of a PD-L1 Blocking Antibody from a Self-Replicating RNA Vector Induces Potent Antitumor Responses

Abstract

Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.

Keywords: AAV; SFV; alphavirus; anti-PD-L1; cancer immunotherapy; colorectal cancer; melanoma; self-replicating RNA.

Figure 1

AAV and SFV Viral Vectors Express Anti-PD-L1 mAb in Cultured Cells and in Tumors In Vivo (A) Schematic representation of AAV and SFV vectors expressing anti-PD-L1 mAb. In SFV-aPDL1, black rectangles at the ends represent 5′ and 3′ viral sequences necessary for replication, and the black arrow represents the SFV subgenomic promoter (the replicase gene is not to scale). (B and C) BHK cells were transfected with AAV-aPDL1 plasmid or infected with the indicated SFV vectors, or mock infected, and the supernatants were collected 48 h later and analyzed as indicated. (B) Western blot was performed under non-reducing (−DTT) or reducing (+DTT) conditions using an anti-mouse IgG peroxidase-conjugated antibody. Red and blue arrows show the complete mAb and the HC of anti-PD-L1 mAb, respectively (LC could not be detected due to low affinity of secondary antibody for lambda LC). (C) A PD-L1-binding ELISA was performed by coating ELISA plates with recombinant PD-L1-Fc and incubating them with serial dilutions of supernatants from cells transfected or infected with the indicated vectors. In order to compare binding of anti-PD-L1 mAb expressed by both vectors, we previously quantified IgG in each sample (using a total mouse IgG ELISA kit) and adjusted mAb concentrations to 40 ng/mL, from which 1:4 dilutions were tested. For the mock-infected sample, the first dilution was prepared with a volume equivalent to the one used for the sample with lower mAb concentration. (D and E) C57BL/6 mice bearing subcutaneous MC38 tumors having an average diameter of 5.5 mm received a single intratumoral dose of 10¹¹ VGs (AAV-aPD-L1), 3 × 10⁸ VPs (SFV-aPD-L1 and SFV-LacZ), or the same volume of saline. The amount of recombinant anti-PD-L1 mAb present in tumor extracts (D) and serum (E) was determined at the indicated times using a PD-L1-binding ELISA (n = 5 for each time point). Asterisks above bars indicate comparison of each group with saline. *p < 0.05; **p < 0.01. One representative experiment out of two performed is shown. 2A, foot-and-mouth disease virus 2A autoprotease; EF1α, human elongation factor 1α promoter; f, furine protease cleavage site; HC, mAb heavy chains; ITR, AAV inverted terminal repeats; LC, mAb light chains; ns, not significant; pA, synthetic polyadenylation sequence. Data represent the mean + SEM.

Figure 2

Evaluation of Antitumor Efficacy of AAV and SFV Viral Vectors Expressing Anti-PD-L1 mAb in MC38 Subcutaneous Tumors A total of 5 × 10⁵ MC38 cells were inoculated subcutaneously into the right flank of C57BL/6 mice and approximately 7 days later (day 0), when the average diameter was 3–3.5 mm, animals received one intratumoral dose of 10⁸ VGs of AAV-aPDL1 or 3 × 10⁸ VPs of SFV-aPDL1or SFV-LacZ (1×). A control group received the same volume of saline. Two additional groups received three intratumoral doses of SFV-aPDL1 or SFV-LacZ given on days 0, 2, and 4 (3×). (A) Evolution of tumor size. Data represent the mean tumor size (mm²) + SEM. (B) Survival after treatment. (C) Survival after tumor rechallenge. Mice treated with SFV-aPDL1 (1× or 3×) that rejected tumors (n = 7) were rechallenged 2–3 months later with 5 × 10⁵ MC38 cells, and survival was analyzed using naive mice that received the same amount of tumor cells as controls (n = 8). For (A) and (B), one representative experiment (n = 7) out of three performed is shown (in B the statistical analysis was performed using pooled data from two independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, not significant.

Figure 3

SFV-aPDL1 Given Intratumorally Showed Greater Antitumor Effect Than Anti-PDL1 mAb Administered Locally or Systemically C57BL/6 mice bearing subcutaneous tumors were inoculated intratumorally with a single dose of SFV-aPDL1 (3 × 10⁸ VPs), as described in Figure 2. In parallel, mice with similar tumors received three intraperitoneal (i.p.) doses of 100 μg of anti-PDL1 mAb on days 0, 3, and 6, or two intratumoral (i.t.) doses of 100 ng of anti-PDL1 mAb on days 0 and 2. Mice treated with saline were used as negative control. (A) Evolution of tumor size (mm²) along time for each individual mouse. The fractions in the right lower corner of each graph indicate the number of complete regressions/total number of mice in each group. Dashed lines indicate the times of mAb administrations. (B) Mean tumor size evolution ± SEM. (C) Survival after treatment (pooled data from two independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. One representative experiment out of two performed is shown. ns, not significant.

Figure 4

Analysis of the Expression of Genes Induced by Type I IFN in MC38 Subcutaneous Tumors C57BL/6 mice bearing subcutaneous MC38 tumors received a single intratumoral dose of 3 × 10⁸ VPs (SFV-aPD-L1 and SFV-LacZ), 10¹¹ VGs (AAV-aPD-L1), or the same volume of saline. Tumor samples were extracted and processed 17 h after treatment as described in the Materials and Methods. Finally, they were analyzed by qRT-PCR with primers specific for the indicated genes. Asterisks above each bar indicate the statistical comparison of each group with the saline group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, not significant. Data represent the mean + SEM.

Figure 5

Analyses of CD8 and CD4 T Cells in MC38 Tumors, Lymph Nodes, and Blood C57BL/6 mice bearing subcutaneous MC38 tumors were inoculated with the indicated vectors or saline as described in Figure 1D. Five days after the administration of vectors, tumors and tumor draining lymph nodes (TDLNs) were excised and digested, and single-cell suspensions were analyzed by flow cytometry. Blood was collected on the same day and analyzed by flow cytometry. Data represent the mean (n = 6) ± SEM of total CD8 (left graphs), tumor-specific CD8 (MC38 Tetr⁺, central graphs), and CD4 T cells (right graphs) in tumors (A), TDLNs (B), and blood (C). Gating strategies are shown in Figure S9. Asterisks above each bar indicate the statistical comparison of each group with the control saline group. Other comparisons are indicated by horizontal bars. ⁺p < 0.1; *p < 0.05; **p < 0.01. ns, not significant.

Figure 6

Analyses of Co-stimulatory and Co-inhibitory Immunological Markers on MC38 Tumor T Cells Tumor-infiltrating lymphocytes obtained as described in Figure 5 were analyzed by flow cytometry with antibodies specific for the immunological co-stimulatory markers CD137 and OX40 (A) and for immunological co-inhibitory markers PD-1 and LAG3 (B). Data show levels (mean ± SEM, n = 6) of each marker in total CD8 T cells (left graphs), tumor-specific CD8 T cells (MC38 Tetr⁺, central graphs), and total CD4 T cells (right graphs). Asterisks above each bar indicate the statistical comparison of each group with the control saline group. Other comparisons are indicated by horizontal bars. *p < 0.05; **p < 0.01; ***p < 0.001. ns, not significant.

Figure 7

Analyses of CD8 and CD4 T Cells in B16-OVA Tumors A total of 5 × 10⁵ B16/OVA cells were inoculated subcutaneously into the right flank of C57BL/6 mice and 6 days later, when tumors had an average diameter of 4–5 mm, animals received one intratumoral dose of 3 × 10⁸ VPs of the indicated SFV-derived vectors or an equivalent volume of saline. Five days after the administration of vectors, tumors were excised, digested, and single-cell suspensions were analyzed by flow cytometry. Data represent the mean (n = 6) ± SEM of total CD8 (left graphs), tumor-specific CD8 (B16-Tetr⁺, central graphs), and CD4 T cells (right graphs) in tumors. Asterisks above each bar indicate the statistical comparison of each group with the control saline group. Other comparisons are indicated by horizontal bars. *p < 0.05; **p < 0.01; ***p < 0.001. ns, not significant.

Figure 8

Treatment Efficacy of SFV-aPDL1 in Combination with Anti-CD137 and Anti-LAG3 mAbs Mice bearing subcutaneous MC38 tumors (4–5 mm of average diameter) were inoculated with one intratumoral dose of SFV-aPDL1 (3 × 10⁸ VPs) or saline (day 0). Then, one third of mice were intraperitoneally injected with anti-CD137 (days 0 and 6) or with anti-LAG3 (days 3, 6, and 9). (A) Graphs show the evolution of tumor size over time for each individual mouse. The fractions in the right lower corner of each graph indicate the number of complete regressions/total number of mice for each group. (B) Data represent the mean tumor size for all groups + SEM (n = 8–9). (C) Survival after treatment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. One representative experiment out of two performed is shown. ns, not significant.