ancer immunotherapies have shown promise in harnessing the immune system to target and destroy cancers, leading to clinical benefit enriched in patients with a high mutational burden [1][2][3][4][5] . Multiple studies indicate that cytotoxic CD8 T cells targeting tumor neoantigens are critical to tumor control and clearance in response to immunotherapies targeting CTLA-4 or PD-1 [6][7][8][9][10] . Clinical responses to CPI therapy rely mostly on reinvigorating preexisting tumor-specific T cell responses 11 , and active vaccination to expand preexisting and prime de novo tumor-specific T cells is anticipated to overcome this limitation.The limited success of cancer vaccines in the past can be attributed to a number of factors, including selection of poorly immunogenic self-antigens 12 , insufficiently immunogenic vaccine platforms and immunosuppressive milieus in patients with advanced cancers 4 . Accordingly, peptide-based neoantigen vaccine platforms have to date failed to consistently induce robust neoantigen-specific CD8 T cell responses in the majority of patients [13][14][15] . Although more immunogenic, a homologous prime boost messenger RNA (mRNA)-based vaccination approach elicited predominantly CD4 T cell responses [16][17][18] . Cumulatively, previous findings suggest that a successful cancer vaccine should (1) target tumor-specific neoantigens, (2) use highly immunogenic vaccine platform(s), (3) expand and prime T cells, (4) be combined with CPI therapy 19 and (5) generate long-term memory responses to ensure continuous tumor control for durable clinical benefit.Viral vector-based vaccine platforms, such as recombinant adenovirus, are able to prime robust T cell responses [20][21][22][23][24] . Although high seroprevalence of anti-adenoviral antibodies in human populations

Purpose: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E7 11-20-specific CD8 þ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. Experimental Design: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. Results: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E7 11-20-specific CD8 þ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E7 11-20-specific CD8 þ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. Conclusions: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E7 11-20-specific population of cytotoxic CD8 þ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPR-ER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The levels of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPR-ER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that cannot be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

<div>AbstractPurpose:<p>To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E7_11-20-specific CD8⁺ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers.</p>Experimental Design:<p>CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas <i>in vivo</i> activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model.</p>Results:<p>CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E7_11-20-specific CD8⁺ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E7_11-20-specific CD8⁺ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy.</p>Conclusions:<p>Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E7_11-20-specific population of cytotoxic CD8⁺ T cells, a favorable safety profile, and <i>in vitro</i> and <i>in vivo</i> evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).</p></div>

<p>Supplementary Table 1</p>

<p>Supplementary Figure 2. Flow cytometric characterization of PBMC expansion cultures.</p>

<p>Supplementary Materials and Methods</p>

<p>Supplementary Figure 1. Characterization of CUE-101 binding properties.</p>