Intricacies for posttranslational tumor-targeted cytokine gene therapy

Abstract

The safest and most effective cytokine therapies require the favorable accumulation of the cytokine in the tumor environment. While direct treatment into the neoplasm is ideal, systemic tumor-targeted therapies will be more feasible. Electroporation-mediated transfection of cytokine plasmid DNA including a tumor-targeting peptide-encoding sequence is one method for obtaining a tumor-targeted cytokine produced by the tumor-bearing patient's tissues. Here, the impact on efficacy of the location of targeting peptide, choice of targeting peptide, tumor histotype, and cytokine utilization are studied in multiple syngeneic murine tumor models. Within the same tumor model, the location of the targeting peptide could either improve or reduce the antitumor effect of interleukin (IL)12 gene treatments, yet in other tumor models the tumor-targeted IL12 plasmid DNAs were equally effective regardless of the peptide location. Similarly, the same targeting peptide that enhances IL12 therapies in one model fails to improve the effect of either IL15 or PF4 for inhibiting tumor growth in the same model. These interesting and sometimes contrasting results highlight both the efficacy and personalization of tumor-targeted cytokine gene therapies while exposing important aspects of these same therapies which must be considered before progressing into approved treatment options.

Figure 1

Expression and activity of IL12 is not affected by insertion of tumor-targeting sequence in either subunit. Multiple VNTANST-IL12 plasmids were created with the VNTANST sequence inserted directly prior to the stop codon in the p40 subunit (a), p35 subunit (b), or both subunits (c). The blue arrows represent the VNTANST-coding sequence insertion site in the plasmid DNA. CMV: cytomegalovirus promoter; IVS: intron; pA: bovine growth hormone polyadenylation signal; STOP: stop codon. (d) Transfection of these plasmids and wild-type IL12 and a control plasmid into cells resulted in equivalent amounts of expressed IL12 in the medium of transfected cells. (e) The peptide IL12 products induced equivalent levels of IFNγ from harvested murine splenocytes. # represents P < 0.05 compared to all other groups.

Figure 2

Location of the tumor-targeting sequence affects the induced antitumor response in a tumor histotype-specific manner. The ttIL12-p40 pDNA treatments increased the antitumor activity compared to the wtIL12 pDNA treatments in the 4T1 ((a) primary tumor growth; (b) lung metastases; (c) survival) and B16F10 ((d) primary tumor growth, (e) survival) tumor models. (f) In the SCCVII model, all ttIL12-peptide pDNA treatments significantly extend survival. Black arrows represent treatment dates. # represents P < 0.05 compared to all other groups. ∗ represents P < 0.05 compared to all other IL12 treatment groups.

Figure 3

Tumor-targeting-mediated improvement of IL15-induced antitumor efficacy depends on the tumor histotype. (a) Diagrammatic representation of the IL15 plasmid with the IL15-coding region in the “Cytokine” region. The blue arrows represent the VNTANST-coding sequence insertion site in the plasmid DNA. CMV: cytomegalovirus promoter; IVS: intron; pA: bovine growth hormone polyadenylation signal; STOP: stop codon. (b) Equivalent inhibition of K7M3 lung metastases from wtIL15 and ttIL15 pDNA treatments (treatments on days 0 and 7). Inhibition of primary tumor growth (c) and metastatic lung tumor development (d) by ttIL15 pDNA compared to wtIL15 pDNA. Black arrows represent treatments. # represents P < 0.05 compared to all other groups.

Figure 4

Tumor-targeting PF4 gene therapy with the VNTANST sequence does not improve anticancer efficacy. Black arrows represent treatments. # represents P < 0.05 compared to all other groups. (a) Only wtPF4 was capable of inhibiting primary tumor growth, but both wtPF4 and ttPF4 equally inhibited the development of lung metastases (b). Black arrows represent treatments. # represents P < 0.05 compared to all other groups.

Figure 5

Not all tumor-targeting peptides can be successfully utilized in this gene delivery method. (a) Multiple tumor-targeting IL12 pDNAs were created by inserting the tumor-targeting peptide-coding sequences into the p40 subunit of the IL12 pDNA as in Figure 1(a). Only the wtIL12, VNTANST-IL12, and CDGRC-IL12 pDNA treatments inhibited primary tumor growth compared to the control pDNA treatments in the 4T1 tumor model. In side-by-side statistical analyses, the inhibition of primary tumor growth with VNTANST-IL12 was significantly inhibited compared to the wtIL12 and CDGRC-IL12 data on day 18 only. (b) Similar results were seen in the development of metastatic tumors in the same mice with only RGD4C not reducing development compared to control. Again, VNTANST-IL12 was significantly different from all groups in side-by-side analyses. Black arrows represent treatments. # represents P < 0.05 compared to control. ∗ represents P < 0.05 compared to all other groups.

Figure 6

Increasing the dose of ttIL12 pDNA abrogates its therapeutic benefits. Increasing the dose by 3-fold to 30 μg pDNA per treatment resulted in a loss of inhibition by ttIL12 pDNA treatments, while the wtDNA treatments retained the ability to inhibit primary tumor growth in 4T1 primary tumor growth. Black arrows represent treatments. # represents P < 0.05 compared to all other groups.