TCR sequencing and analysis of stable T-cell clonesĪll animal studies followed guidance from the SmartLabs Institutional Animal Care and Use Committee protocol MIL-100 and were performed in compliance with federal guidelines. Cells were next washed in permeabilization buffer (eBioscience), stained with intracellular antibodies for 30 minutes at room temperature, washed, and analyzed. Cells were stimulated for 5 hours, washed, stained with FVS780 and surface markers as above, and fixed using IC fixation buffer (Thermo Fisher Scientific). Expanded PBMCs or murine cells treated with media containing BFA and Monensin or Cell Stimulation Cocktail (Thermo Fisher Scientific) served as controls. ![]() Alternatively, 2 × 10 6 murine cells were plated in a 96-well U-bottom plate and restimulated in CTL Test Medium containing 1% Glutamax by combining E7 49-57 peptide (RAHYNIVTF 10 μg/mL) with BFA and Monensin diluted per the manufacturer's instructions. Materials and MethodsĪ total of 2 to 4 × 10 6 human PBMCs expanded with CUE-101 or peptide were pretreated with Brefeldin A (BFA) and Monensin (Thermofisher), plated in a 24-well plate, and stimulated at a 1:1 ratio with T2 cells (ATCC) that had been loaded with 5 μg/mL of E7 11-20 (YMLDLQPETT) or HIV-1 p17 Gag 77-85 (SLYNTVATL SL9) peptide for 2 hours and washed twice. CUE-101 is currently being evaluated in patients with HPV16 + HNSCC (NCT03978689). ![]() These data support the potential for CUE-101 to enhance antitumor immunity in HPV16-driven malignancies. This activity is observed in vitro in primary human cells and in vivo in HLA-A2 transgenic mice, and the therapeutic relevance of this mechanism is further supported by a murine surrogate (mCUE-101) that expands sufficient E7-specific CD8 + T cells to promote antitumor efficacy and induce functional immunologic memory in the TC-1 tumor model. As the first example in this molecular series, CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E7 11-20-specific CD8 + cytotoxic T cells, consistent with its design. Together, the selective engagement and expansion of tumor antigen-specific T cells suggest potential for anticancer efficacy with reduced toxicity relative to nontargeted forms of immunotherapy that result in systemic activation of the immune system. The novel mechanism of action of these fusion proteins harnesses two key signals for T-cell activation: T-cell receptor (TCR) engagement of a tumor-associated HLA-A*0201-peptide complex to induce activation, and delivery of affinity-attenuated IL2 to induce proliferation of tumor antigen-specific T cells. The Immuno-STAT™ (Selective Targeting and Alteration of T cells) platform utilizes a highly modular molecular framework to directly modulate the activity of antigen-specific T cells in vivo. In vivo studies confirm selective expansion of tumor-specific cytotoxic CD8 + T cells, induction of immunologic memory, and a favorable safety profile, suggesting the potential for clinical efficacy in patients with HPV16 + malignancies. ![]() CUE-101 is a novel fusion protein that demonstrates selective binding, activation, and expansion of HPV16 E7 11-20-specific CD8 + T cells in preclinical studies. The observation of objective clinical responses in early trials testing adoptive cell therapy with gene-engineered T cells targeting HPV16 E7 supports the concept that a single T-cell specificity can lead to antitumor activity in patients with HPV16-driven cancers. Despite recent approvals of immunotherapy drugs for the treatment of HNSCC and the limited clinical success that has been reported with therapeutic vaccines and CAR-T and adoptive cell therapies, metastatic disease remains largely incurable. Human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is emerging as a global epidemic.
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