Topical Delivery of Nivolumab, a Therapeutic Antibody, by Fractional Laser and Pneumatic Injection.

BACKGROUND AND OBJECTIVES: PD-L1 is a tumor ligand that binds to the PD-1 receptor on immune cells, thereby inhibiting the antitumor immune response. The antibody nivolumab is a PD-1 inhibitor, Food and Drug Administration approved for systemic treatment of several aggressive cancer types. Topically applied nivolumab may hold potential as a future strategy to treat keratinocyte cancer, but its molecular properties preclude unassisted topical uptake. The aim of this study was to investigate uptake and biodistribution of topically delivered nivolumab, assisted by two physical enhancement techniques with different delivery kinetics; ablative fractional laser (AFL) and electronically controlled pneumatic injection (EPI). STUDY DESIGN/MATERIALS AND METHODS: In vitro porcine skin was exposed to CO2 AFL (20 mJ/mb, 5% density), followed by passive diffusion of nivolumab in a Franz cell (1 mg/ml, 18 hours, n = 6) or treated with EPI (4 bar) for immediate delivery of nivolumab (1 mg/ml, 10 minutes, n = 6). The resulting nivolumab skin concentrations were quantified by enzyme-linked immunosorbent assay (ELISA) at three skin depths (100, 500, and 1500 µm), comparing the uptake from assisted delivery with intact skin. Biodistribution of nivolumab in the skin for all interventions was visualized by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and fluorescence microscopy. RESULTS: Delivery of nivolumab by AFL-assisted passive diffusion and immediate EPI both resulted in significantly enhanced uptake of nivolumab in all skin depths compared with intact skin (P < 0.05). With AFL, nivolumab concentrations reached 86.3 µg/cm3 (100 µm), 105.8 µg/cm3 (500 µm), and 19.3 µg/cm3 (1500 µm), corresponding to 2-10% of the applied concentration, with the highest deposition in the mid dermis. Immediate EPI delivered 429.4 µg/cm3 (100 µm), 584.9 µg/cm3 (500 µm), and 295.9 µg/cm3 (1500 µm) into the skin, corresponding to 29-58% of the applied nivolumab concentration. From qualitative visualization of the biodistribution, it appeared that nivolumab distributed in a horizontal and continuous homogenous band in the upper and mid dermis through AFL-exposed skin, whereas EPI-delivery showed a deep focal deposition extending into the deep dermis. CONCLUSIONS: AFL-assisted passive diffusion and immediate EPI-assisted delivery show the potential to deliver therapeutic antibodies locally. Future in vivo and pharmacokinetic studies would reveal the full potential for topical antibody delivery by energy-based devices. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

High diversity of the T-cell receptor repertoire of tumor-infiltrating lymphocytes in basal cell carcinoma.

Whether specific T-cell clones are present in tumor infiltrating lymphocytes (TILs) in BCC is unknown. We employed deep sequencing of mRNA coding for the T-cell receptor (TCR) chains α- and ß to characterize the repertoire of TILs in BCC. V and J gene-usage and CDR3 length were computed to determine the clonality of TCR and degree of overlap in TCR repertoires between skin resident T-cells and TILs. We found high diversity of the TCR repertoire in BCC and control skin with random V-J gene usage and similar CDR3-length distribution. Lack of TCR repertoire restriction indicates absence of tumor-specific TIL clones in BCC.

Immunosuppressive Environment in Basal Cell Carcinoma: The Role of Regulatory T Cells.

Interaction between tumour survival tactics and anti-tumour immune response is a major determinant for cancer growth. Regulatory T cells (T-regs) contribute to tumour immune escape, but their role in basal cell carcinoma (BCC) is not understood. The fraction of T-regs among T cells was analysed by immunohistochemistry followed by automated image analysis in facial BCC, peritumoural skin and normal, buttock skin. Quantitative real-time PCR (qRT-PCR) was performed for Foxp3 and cytokines involved in T-reg attraction and T-cell activation. T-regs comprised 45% of CD4-cells surrounding BCC. Foxp3 was highly expressed in BCC, but absent in buttock skin. Unexpectedly, expression of Foxp3 was increased in peritumoural skin, with the FoxP3/CD3 fractions exceeding those of BCC (p = 0.0065). Transforming growth factor (TGF)-ß and T-reg chemokine expression was increased in BCC and peritumoural skin, but not in buttock skin, with expression levels correlating with Foxp3. T-regs are abundantly present both in BCC and in peritumoural skin, mediating an immunosuppressed microenvironment permissive for skin cancer