In situ vaccination against mycosis fungoides by intratumoral injection of a TLR9 agonist combined with radiation: a phase 1/2 study

Abstract

We have developed and previously reported on a therapeutic vaccination strategy for indolent B-cell lymphoma that combines local radiation to enhance tumor immunogenicity with the injection into the tumor of a TLR9 agonist. As a result, antitumor CD8(+) T cells are induced, and systemic tumor regression was documented. Because the vaccination occurs in situ, there is no need to manufacture a vaccine product. We have now explored this strategy in a second disease: mycosis fungoides (MF). We treated 15 patients. Clinical responses were assessed at the distant, untreated sites as a measure of systemic antitumor activity. Five clinically meaningful responses were observed. The procedure was well tolerated and adverse effects consisted mostly of mild and transient injection site or flu-like symptoms. The immunized sites showed a significant reduction of CD25(+), Foxp3(+) T cells that could be either MF cells or tissue regulatory T cells and a similar reduction in S100(+), CD1a(+) dendritic cells. There was a trend toward greater reduction of CD25(+) T cells and skin dendritic cells in clinical responders versus nonresponders. Our in situ vaccination strategy is feasible also in MF and the clinical responses that occurred in a subset of patients warrant further study with modifications to augment these therapeutic effects. This study is registered at www.clinicaltrials.gov as NCT00226993.

Figure 1

Treatment schema. The single immunization procedure (A) consisted of irradiating a single MF skin lesion on days 1 and 2 (2 Gy each day) along with intratumoral injection of CpG within an hour before the day 1 and after the day 2 RT. CpG was then injected into the same site weekly for an additional 8 doses for a total of 10 doses. From patient 7 (B), a boost immunization was delivered at a distant MF lesion at week 4, and weekly CpG injections were performed at that site until completion of 10 doses. Systemic clinical response was assessed by mSWAT of nonimmunized MF lesions weekly until week 8 and then at week 12. Correlative skin samples were obtained from the immunization site at baseline and week 2.

Figure 2

Waterfall plot showing percent change in skin mSWAT score at time of best clinical response. All values are shown as the percentage of change in the mSWAT score compared with the baseline assessment calculated at screening.

Figure 3

Percentage of change in mSWAT score from baseline (skin tumor burden). (A) Single immunization schedule, patients 1 to 6. (B) Dual immunization schedule, patients 7 to 15.

Figure 4

Systemic antitumor clinical response observed with single or dual immunization schedule. (A) Patient 2 (64-year-old man, stage IIB) was treated with the single immunization procedure. Systemic evaluation site was the left calf, and the immunization site was the left thigh. PR was noted at week 8, with DOR of 12 weeks. Patient met progression criteria per mSWAT score at week 20 and withdrew from the trial to start topical steroid therapy. (B) Patient 9 (71-year-old man, stage IB) received dual immunization treatment. Systemic evaluation sites were the lower legs, and lesions on each thigh served as the immunization sites. PR was noted at week 12, with DOR of 6 weeks. Patient withdrew from trial at week 18 to initiate topical nitrogen mustard to small areas of recurrent skin disease.

Figure 5

Significant decreases in CD25⁺, FoxP3⁺ T cells and APCs, whereas CD123⁺ pDCs show increases at the immunized site. (A) A significant decrease in the CD25, FoxP3–expressing T-cell infiltrate was noted when comparing skin biopsies taken from the immunization site before and after treatment (week 2). (B) S100⁺ and CD1a⁺ DC numbers also decreased at these time points, whereas CD123⁺ pDC numbers increased. Images were acquired using a model BX51 microscope with a 100×/1.25 NA Plan oil objective lens and Microfire digital camera with PictureFrame Version 2.3 software (Olympus). Digitized images were processed using Photoshop 7 image processing and manipulation software (Adobe Systems).

Figure 6

Dose-responsive activation of peripheral blood pDCs and B-cells by CpG. These are dose-response histograms showing activation of pDCs and B cells (measured by expression of CD83) with incremental amounts of CpG. In this study, leukocytes were isolated from heparinized peripheral blood from patients or healthy controls by Ficoll gradient and cultured in complete media at 37°C, 5% CO₂ for 18 hours with 0, 3, or 10 μg/mL CpG ODN PF-3512676. Afterward, cells were washed and stained with the following antibody panel: CD3, CD14, CD16, CD56 (lin)–FITC, CD83-PE, CD123-PE-Cy7, HLA-DR-allophycocyanin, and CD19-AMCyan. Then, the sensitivity was assessed on an LSR-II flow cytometer. The responsiveness of TLR9 to CpG was similar in MF compared with healthy controls in both pDCs and B cells.