Discovery of a linear peptide for improving tumor targeting of gene products and treatment of distal tumors by IL-12 gene therapy

Abstract

Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments.

Figure 1

Accumulation of peptide-SEAP reporter gene products and CHP-biotin in tumors. (a) The peptide-SEAP constructs with insertion of the peptide-coding sequence directly before the stop codon (blue arrow). (b) T/S SEAP levels 72 hours after intramuscular EP of peptide-SEAP plasmid DNA in syngeneic CT26 (orange, n = 3), SCCVII (red, n = 4), AT84 (blue, n = 4), and 4T1 (green, n = 4) tumor-bearing mice as well as xenogeneic MCF7 tumor-bearing mice (purple, n = 4). Columns represent the ratio of the control-normalized SEAP/protein (pg/mg) in tumor to SEAP (pg/ml) in the serum and error bars represent SEM (*P < 0.05 compared to wtSEAP). (c,d) DAB staining of tumor tissues from CHP-biotin and control-peptide-biotin treated mice counterstained with either hematoxylin (blue) or eosin (pink). The bottom images are larger versions of the areas within the white squares. Bar = 100 µm in the top panels and bar = 200 µm in the bottom panels. CHP, carcinoma homing peptide; CMV, cytomegalovirus promoter; EP, electroporation; IVS, intron; pA, bovine growth hormone polyadenylation signal; SEAP, secreted alkaline phosphatase-coding sequence; STOP, stop codon.

Figure 2

Biological activity and targeting ability of peptide-IL-12 fusion gene products. (a) The peptide-IL-12 constructs with insertion of the peptide-coding sequence directly before the stop codon in the p40 coding region (blue arrow). (b) Expression of interleukin-12 (IL-12) after in vitro transfection of 4T1 cells with control, wtIL-12, and peptide-IL-12 (n = 3) and (b) induction of interferon-γ (IFN-γ) from splenocytes after transfer of condition medium containing control, wild-type IL-12 (wtIL-12), or peptide-IL-12 gene products. (d) IL-12 accumulation in tumor-bearing IL-12^−/− mice treated with carcinoma homing peptide (CHP)-IL-12 or wtIL-12 determined via an IL-12p70 enzyme-linked immunosorbent assay. Columns represent the wtIL-12-normalized level of IL-12/protein (pg/mg) in tumor per IL-12/protein (pg/mg) in kidneys (green), livers (blue), and spleens (red) and IL-12 pg/ml serum (orange, n = 4). Error bars represent SEM (*P < 0.05 compared to all groups). CMV, cytomegalovirus promoter; IVS, intron; pA, bovine growth hormone polyadenylation signal; SEAP, secreted alkaline phosphatase-coding sequence; STOP, stop codon.

Figure 3

Inhibition or regression of primary tumor growth, decrease in metastatic tumor incidence, and increase in survival time by systemic delivery of CHP-IL-12 plasmid DNA. (a) Tumor growth following treatments with CHP-IL-12, wild-type IL-12 (wtIL-12), and control plasmid DNA in 4T1 tumor-bearing balb/c mice (n = 5; *P < 0.05 at day 30 and P < 0.001 from day 33 until day 42 compared to wtIL-12 plasmid DNA and P < 0.01 at day 21 and P < 0.001 from day 24 to day 33 compared to control plasmid DNA). (b) Metastatic nodules in the lungs of mice (n = 5) treated as in (a) and killed 17 days after the second treatment (*P < 0.05 compared to wtIL-12 plasmid DNA; #P < 0.001 compared to control plasmid DNA). (c) Kaplan–Meier survival analysis of the same mice treated in (a) (*P < 0.05 compared to wtIL-12 plasmid DNA; #P < 0.001 compared to control plasmid DNA). (d) Tumor growth following treatments as in a in SCCVII tumor-bearing C3H mice (n = 5; *P < 0.05 on days 17 and 20 compared to wtIL-12 plasmid DNA and control plasmid DNA). (e) Kaplan–Meier survival analysis of the same mice treated in (d) (*P < 0.05 compared to wtIL-12 and control plasmid DNA). (f) Treatments as in a in CT26 tumor-bearing balb/c mice (n = 5; *P < 0.05 compared to wtIL-12 plasmid DNA, n = 4, on day 25, and control plasmid DNA, n = 3, on days 19 through 25). Black arrows represent treatments, and error bars represent SEM. CHP, carcinoma homing peptide; IL-12, interleukin-12.

Figure 4

Increased antitumor immune response by CHP-IL-12 fusion gene treatments. (a) Fluorescence-activated cell sorting analysis of tumor-infiltrating cells isolated from SCCVII tumors from C3H mice treated as described in Figure 5 collected 7 days after the second treatment. The top right quadrant of the colored dot plot representation of cells gated for CD11c⁺ represents activated dendritic cells (CD80^hi). (b) Tumor-specific cytotoxic T lymphocytes (CTL) activity from wtIL-12 and CHP-IL-12 fusion gene plasmid DNA treated mice bearing orthotopic EMT6 tumors collected (*P <0.05). (c) Serum interferon-γ (IFN-γ) levels from 4T1-tumor bearing Balb/c 3 days after treatments with CHP-IL-12, wtIL-12, and control plasmid. Error bars represent SEM. CHP, carcinoma homing peptide; IL-12, interleukin-12; wtIL-12, wild-type IL-12.

Figure 5

Identification of carcinoma homing peptide (CHP) interacting protein: vimentin. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of potential receptors for CHP isolated via affinity chromatography of a pool of cell-surface proteins isolated from SCCVII cells. The only distinct band (white arrow) was located in the second fraction, and mass spectrometry identified this band as vimentin. (b) Interaction of CHP-biotin with recombinant vimentin-GST (Vimentin), GST, and coating buffer only (control) coated wells of a polystyrene plate (n = 6; *P < 0.001 compared to both GST and control, errors bars represent SEM). (c) Western blot analysis of vimentin expression in an (1) SCCVII tumor and (2) heart, (3) lung, (4) liver, (5) kidney, (6) spleen, and (7) serum from SCCVII-tumor bearing C3H mice. (d) Western blot analysis of vimentin expression in in vitro and ex vivo tumor samples. (e) Accumulation of peptide-biotin in syngeneic SCCVII tumor-bearing C3H mice following intravenous (i.v.) injection of either control-biotin (top left and right) or CHP-biotin (bottom left and right) without (top and bottom left) or with (top and bottom right) depletion of vimentin with a coinjection of purified polyclonal goat antivimentin (100 µg) in the same i.v. injection as the peptide-biotin. BSA, bovine serum albumin; GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

Figure 6

Decreased liver toxicity of interleukin-12 (IL-12) treatments with carcinoma homing peptide (CHP)-IL-12. (a) Number of SCCVII tumor-bearing C3H mice with toxic lesions on the liver following two treatments of 1 µg (2 × 1 µg), 2 µg (2 × 2 µg), or 10 µg (2 × 10 µg) or three treatments of 2 µg (3 × 2 µg) of wild-type IL-12 (wtIL-12) or CHP-IL-12 (n = 12). (b) Representative image of a normal liver area. (c) Representative image of a toxic lesion. Bar = 50 µm in (b) and (c). (d) Levels of alanine transaminase (ALT), a key indicator of liver function, for both plasmid DNA treatments at all DNA levels and time points.