Purpose:

To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-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.

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 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-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.

Conclusions:

Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ 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).

This article is featured in Highlights of This Issue, p. 1779

Translational Relevance

Human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is emerging as a global epidemic. 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. 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. CUE-101 is a novel fusion protein that demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells in preclinical studies. 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.

Oncogenic human papilloma virus (HPV) is responsible for many cervical and anal cancers and head and neck squamous cell carcinoma (HNSCC; refs. 1, 2). Approximately 70% to 80% of HPV-driven oropharyngeal cancers in the United States are HPV16/18 driven, and their incidence continues to rise (3, 4). Prophylactic HPV vaccines have no therapeutic effect on established disease, thus HPV infections are expected to continue contributing to the global cancer burden (5). Recent studies suggest that HPV+ cancers may be successfully targeted with T-cell therapy, wherein adoptive transfer of tumor-infiltrating or genetically engineered T cells was shown to induce responses in HPV-associated cancer patients (6–8). These studies provide proof of concept that a therapeutic strategy that increases the frequency of tumor antigen specific T cells may be sufficient to drive clinical benefit in this population.

HPV16 E7 is constitutively expressed in HPV-associated cancers, is necessary for both initiation and maintenance of malignant transformation, and is genetically conserved in cancer with essentially no variation in sequence among isolates of precancerous and cancerous lesions in subjects from around the world (9). Importantly, the E711-20 peptide is maintained in cancer, is an immunodominant CD8 epitope in humans, and is one of the E7 epitopes identified with relatively high affinities for HLA-A*0201, which is expressed in approximately 40% to 50% of people of European descent (10–12). Although the E7 oncoprotein is a promising target for therapy of HPV-driven cancers, HPV driver oncoprotein-directed vaccine therapy has thus far been unsuccessful in patients with metastatic HPV+ cancers (13–16).

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. 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. 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. As the first example in this molecular series, CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ cytotoxic T cells, consistent with its design. 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. These data support the potential for CUE-101 to enhance antitumor immunity in HPV16-driven malignancies. CUE-101 is currently being evaluated in patients with HPV16+ HNSCC (NCT03978689).

Manufacturing, purification, and characterization of Immuno-STAT proteins

Immuno-STAT proteins were expressed in CHO cells, either transiently in Expi-CHO cells (Thermo Fisher Scientific) or in stably producing lines. In general, proteins were purified from conditioned media using a two-step method of Protein A capture with MabSelect SuRe (GE) followed by size exclusion chromatography. Alternatively, the protein was purified using a three-step method, also employing MabSelect SuRe as a first step, followed by anion exchange chromatography and a hydroxyapatite polishing step.

Binding measurements were performed using bio-layer interferometry (Octet RED96e; ForteBio) with antihuman IgG Fc capture biosensor tips (Molecular Devices) to immobilize IL2Rα-Fc (Sino Biological) or IL2Rβ-Fc (Acro Biosystems). Interactions with anti-HLA (clone W6/32; Abcam) were measured by capturing CUE-101 and analogs on biosensor tips. Raw data were processed and analyzed with ForteBio's Data Analysis 11.0 software. For kinetic measurements, the processed curves were globally fit to a 1:1 binding model (17).

CTLL2 proliferation assay

CTLL2 cells (ATCC) were cultured in complete RPMI supplemented with IL2 (50 IU/mL). One day prior to testing, cells were washed with culture medium without IL2, resuspended at 1 × 105 cells/mL, and incubated for 24 hours at 37°C. Test article was then added, and after 3 days of treatment cell viability was assessed by measuring ATP levels via bioluminescence (CellTiter-Glo; Promega) according to manufacturer's instructions.

Cellular binding and phosphoflow analysis

E7 and CMV antigen-specific CD8+ T cells (Astarte Biologics) were thawed, washed, and rested at 37°C. Cells were incubated with Immuno-STAT on ice for 5 minutes and binding was detected with an anti-human-IgG1 Fc-fluorescein isothiocyanate antibody (Abcam) for 15 minutes. For phosphoflow studies, cells were stimulated for 2 minutes (pSLP76) or 5 minutes (pSTAT5) at 37°C. Positive control stimulations included cross-linking anti-CD3/anti-CD28/anti-CD8 antibodies or recombinant human IL2. Cells were immediately fixed, permeabilized, and stained with anti-pY128 SLP76 or anti-pY694 STAT5 antibodies (BD Biosciences) for analysis on an Attune NxT flow cytometer (Invitrogen). The percent of positively stained cells was determined using FlowJo software (TreeStar).

ELISpots

Cells were thawed, washed, and resuspended in CTL-Test media (CTL) supplemented with 1% Glutamax (Gibco). E7 or CMV-specific cells were seeded at a density of 1,000 cells/well with media, Immuno-STAT, or PMA/ionomycin and incubated for 20 hours at 37°C in single enzymatic hu-IFNγ 96-well ELISpot plates (CTL). Plates were developed according to the manufacturer's instructions prior to signal quantitation (Immunospot S6 Universal Analyzer; CTL).

In vitro T-cell expansion

Human healthy donor peripheral blood mononuclear cells (PBMC) were obtained as frozen stocks (Astarte Biologics) or isolated from leukopaks (HemaCare). PBMCs were stimulated with CUE-101 in ImmunoCult-XF T Cell Expansion Media (Stemcell Technologies) for 10 days at 37°C with 50% media replacement every 2 to 3 days starting on day 5. As a control, PBMCs were stimulated for 10 days with 5 μg/mL of E711-20 peptide (Elim Biopharma) and IL2 (50 U/mL), with 50% media and IL2 replaced every 2 to 3 days starting on day 3. After 10 days, the cells were harvested and stained for downstream assays.

Tetramer staining and flow cytometry analysis

Prior to tetramer staining, murine cells were resuspended in 50 nmol/L dasatinib and incubated for 30 minutes at 37°C. Cells were washed, resuspended in Fixable Viability Stain 780 (BD Biosciences), and incubated for 15 minutes at 4°C. Cells were then pelleted and stained with tetramer (MBL International) diluted 1:20 in FACS buffer for 15 minutes at room temperature followed by surface staining for 20 minutes at 4°C. Fc receptor binding inhibitor (eBioscience) or 0.5 μg/mL mouse Fc Block (Clone 2.4G2; BD Biosciences) was also included. Cells were washed and fixed for 30 minutes at 4°C using the FoxP3 Fixation/Permeabilization Kit (eBioscience), washed, stained for FoxP3 for 30 minutes at room temperature, and analyzed on an Attune NxT cytometer.

Intracellular cytokine staining

A total of 2 to 4 × 106 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 E711-20 (YMLDLQPETT) or HIV-1 p17 Gag77-85 (SLYNTVATL; SL9) peptide for 2 hours and washed twice. Alternatively, 2 × 106 murine cells were plated in a 96-well U-bottom plate and restimulated in CTL Test Medium containing 1% Glutamax by combining E749-57 peptide (RAHYNIVTF; 10 μg/mL) with BFA and Monensin diluted per the manufacturer's instructions. Expanded PBMCs or murine cells treated with media containing BFA and Monensin or Cell Stimulation Cocktail (Thermo Fisher Scientific) served as controls. Cells were stimulated for 5 hours, washed, stained with FVS780 and surface markers as above, and fixed using IC fixation buffer (Thermo Fisher Scientific). Cells were next washed in permeabilization buffer (eBioscience), stained with intracellular antibodies for 30 minutes at room temperature, washed, and analyzed.

TCR sequencing and analysis of stable T-cell clones

PBMCs from healthy donors were expanded in vitro with E711-20 peptide plus IL2, or with 100 nmol/L CUE-101 in 10-day cultures. Expanded cells were harvested, tetramer stained, and E711-20-specific CD8+ T cells were single cell sorted (Sony SH800) and the α and β TCR chains were coamplified (iRepertoire). TCR library sequencing used an Illumina MiSeq v2 Nano Kit. Data for each individual well were demultiplexed, mapped, and analyzed using the iRmap VDJ pipeline and the iPair Analyzer.

SKW-3 cells (DSMZ) were cultured in complete RPMI1640 without pen-strep. TCR lentiviral construct design followed the method of Jin and colleagues (18), and the generation of stable TCR-expressing SKW-3 cells is described in the Supplementary Materials and Methods. Assessment of CD69 upregulation was performed by loading T2 cells with peptide and coincubating with stable TCR transduced SKW-3 cells overnight at 37°C. The cells were stained for CD3, CD19, live/dead, and CD69 in the presence of Fc receptor inhibition, and analyzed.

Animals and tumor studies

All animal studies followed guidance from the SmartLabs Institutional Animal Care and Use Committee protocol MIL-100 and were performed in compliance with federal guidelines. Animal welfare was monitored daily and animal weights were collected twice a week. Early euthanasia criteria included >20% weight loss, body condition score ≤2, or tumor size of >20 mm on the longest side. Caliper measurements to calculate tumor volume (L × L × W/2) were collected three times per week. TC-1 cells were obtained from the laboratory of Dr. TC Wu (Johns Hopkins University) and were authenticated by IDEXX Cell Check Plus (not shown). For tumor studies, age-matched female albino C57Bl/6 (C57Bl/6-Tyrp1b-J/J; Taconic) mice were implanted subcutaneously with 1.5 × 105 TC-1 cells suspended in PBS. For immunization studies, HLA-A2 (B6.Cg-Immp2lTg(HLA-A/H2-D)2Enge/J; Jackson Laboratories) mice were injected subcutaneously with E711-20 peptide, Tetanus Toxin Universal Helper Epitope TT830-844 (QYIKANSKFIGITEL), and CpG ODN1826 (5′-tccatgacgttcctgacgtt-3′) diluted in 1× PBS. Preparation of tissue samples for analysis is described in Supplementary Materials and Methods.

Statistical analysis

Comparisons between two groups were performed by two-tailed Student t tests. For comparisons between multiple groups, one-way ANOVA followed by Tukey post hoc comparison was performed. P values ≤0.05 were considered significant. Comparisons of overall survival were conducted by log-rank test. All analyses were performed using GraphPad Prism (v8.0).

Design and characterization of CUE-101

CUE-101 is composed of a single IgG1 Fc, two peptide–HLA complexes, and four reduced affinity human IL2 domains encoded in a two plasmid system (Fig. 1A and B). By presenting an immunodominant peptide epitope derived from the HPV16 E7 protein (amino acid residues 11-20: YMLDLQPETT), the CUE-101 molecule was designed to target HPV16 E711-20-specific CD8+ T cells for the treatment of HLA-A*0201 patients with HPV16-positive malignancies. Two amino acid substitutions (L234A; L235A) were introduced in the Fc region of CUE-101 to reduce Fc domain effector function (19), allowing CUE-101 to maintain binding to FcRn while not exhibiting measurable binding to other Fc receptors or to C1q, except for FcRI (Supplementary Table 1; Supplementary Fig. S1A). Native conformation of the peptide-HLA component of CUE-101 was confirmed via binding of an anti-HLA class I mAb (Clone W6/32), which recognizes a conformational epitope encompassing the α3, α2, and β2-microglobulin domains (refs. 20, 21; Supplementary Fig. S1B). Analogs of CUE-101, namely CUE-1380-839 (containing CMV pp65495-503 peptide) and CUE-1644-1274 (containing E711-20 peptide but lacking IL2 domains), showed similar binding to this antibody whereas the murine surrogate mCUE-101 (with murine IgG2a Fc and murine H-2Db-E749-57 complexes) did not show significant binding (Supplementary Figs. S1C–S1E). Together, these data demonstrate effector attenuation of the Fc and confirm that the peptide–HLA component of CUE-101 is intact and properly folded.

To increase selectivity for target T cells and to minimize the potential for toxicity mediated through global IL2-driven activation of IL2 receptor (IL2R) expressing cells, two point mutations (H16A and F42A) were introduced into the IL2 sequences of CUE-101. Mutation of F42A was previously demonstrated to reduce IL2 interaction with the IL2Rα (IL2RA) chain (22), whereas H16A was hypothesized to reduce binding to the IL2Rβ (IL2RB) subunit based on structural analyses (23). As predicted, the binding affinity of a double mutant IL2 (H16A; F42A) for human IL2Rα and IL2Rβ was decreased 110- and 3-fold, respectively, compared with wild-type (WT) IL2 binding, predominantly due to a faster off-rate for each of these interactions (Table 1).

Functional attenuation of the mutant IL2 components of CUE-101 was shown in a CTLL-2 proliferation assay (Fig. 1C). Although CUE-101 stimulated proliferation of CTLL-2 cells, the potency of CUE-101 (EC50 = 26.56 nmol/L) was reduced ∼2,600-fold relative to recombinant human IL2, aldesleukin (EC50 = 0.01 nmol/L), confirming both the functionality of the mutant IL2 components of CUE-101 and their marked functional attenuation in an assay that is independent of TCR engagement. This finding supports the potential for CUE-101 to selectively activate antigen specific T cells in the absence of global stimulation of IL2R expressing cells.

CUE-101 selectively binds and activates E711-20-specific CD8+ T cells through both the TCR and IL2R

Only HPV16 E711-20-specific CD8+ T cells express both the TCR and IL2R necessary for full engagement of CUE-101, although immune cell lineages including CD8+ and CD4+ T cells, regulatory T cells, and NK cells could engage CUE-101 via the IL-2R. Selective binding of CUE-101 to HPV16 E711-20-specific CD8+ T cells was demonstrated, whereas only minimal CUE-101 binding to CMV pp65495-503-specific CD8+ T cells, naïve CD8+ T cells, and NK cells was observed (Fig. 1D; Supplementary Fig. S1F). CUE-101 binding induced phosphorylation of tyrosine 128 of the SH2 Domain-Containing Leukocyte Protein of 76 kDa (SLP76; LCP2) and phosphorylation of tyrosine 694 of the STAT5 protein, which serve as readouts for CUE-101 engagement of the TCR (24) and IL-2R (25), respectively. CUE-101 selectively induced SLP76 phosphorylation in HPV16 E711-20-specific CD8+ T cells but not in bulk CD8+ T cells from a healthy donor or in pp65495-503-specific CD8+ T cells (Fig. 1E). Likewise, CUE-101 induced potent and dose-dependent STAT5 phosphorylation in HPV16 E711-20-specific CD8+ T cells (Fig. 1F). Together these results support that the peptide-HLA moiety of CUE-101 directs the selective binding and activation of TCR and IL2R signaling events in antigen-specific T cells.

In addition to cellular binding and the observed signaling events, CUE-101 also induced potent and dose-dependent secretion of IFNγ (IFNG), a T-cell effector cytokine, from primary purified human E711-20-specific T cells (Fig. 1G and H). Removal of the IL2 domains in CUE-1644-1274 significantly reduced the potency of effector cell stimulation (Supplementary Fig. S1G). In contrast, CUE-101 treatment did not activate CMV pp65495-503-specific T cells (Fig. 1G and H). Through substitution with the pp65495-503 peptide, a CMV-specific Immuno-STAT (CUE-1380-839) was generated that specifically activated pp65495-503-specific T cells but not E711-20-specific T cells (Fig. 1G and I). These findings demonstrate that the peptide-HLA moiety of Immuno-STAT molecules guides selective activation of antigen-specific T cells.

CUE-101 selectively expands polyfunctional E711-20-specific CD8+ T cells from human PBMCs

To demonstrate that CUE-101 can enhance antitumor immunity through the selective expansion of E711-20-specific T cells, PBMCs from a healthy HLA-A*0201-positive donor were stimulated with CUE-101, incubated for 10 days, and analyzed by flow cytometry after staining with HLA-A*0201 tetramers presenting the HPV16 E711-20 peptide (Fig. 2A; Supplementary Fig. S2A). Treatment with CUE-101 alone induced a concentration-dependent and selective expansion of E711-20-specific CD8+ T cells from undetectable frequencies at baseline, with maximal expansion observed upon stimulation with 100 nmol/L CUE-101 (Fig. 2B). Similar expansion was observed in PBMCs from a second donor (Supplementary Fig. S2B). CUE-101 also induced expansion of NK cells with minimal or no expansion of CD4+ T cells, total CD8+ T cells, or regulatory T cells (Fig. 2B).

Functionality of the CUE-101 expanded E711-20-specific CD8+ T cells was demonstrated by measuring production of effector cytokines and markers of degranulation in response to stimulation by peptide-pulsed T2 cells (Supplementary Fig. S2C). The CUE-101 expanded tetramer positive T cells stimulated with E711-20-loaded cells demonstrated strong induction of IFNγ, TNFα (TNF), and CD107a (LAMP1; Fig. 2C). These markers were not induced upon stimulation with irrelevant HIV Gag77-85 (SL9)-loaded cells, nor in non-antigen–specific CD8+ T cells stimulated with either peptide (Fig. 2D and E). These results confirm the antigen specificity of the CUE-101 expanded T cells and provide evidence of multiple CTL-like effector functions in response to target cells. TCR repertoire diversity was examined by single-cell TCRα-chain and TCRβ-chain sequencing of tetramer+ CD8+ T cells that were expanded from PBMCs of two healthy donors via CUE-101 or peptide/IL2 stimulation. Although the CD8+ T cells expanded from one donor represented a single TCR specificity, the second donor displayed multiple unique TCR clones, demonstrating that CUE-101, like native peptide, is able to stimulate a polyclonal population of TCRs specific for the HPV16 E711-20 peptide (Fig. 2F). Finally, the two dominant TCRs identified in this analysis were constitutively expressed in SKW-3 cells and were shown to be responsive to both E711-20 and E711-19 peptide stimulation (Fig. 2G), demonstrating that CUE-101 expands effector CD8+ T cells capable of responding to both of these immunodominant peptide epitopes (10, 11).

CUE-101 treatment expands E711-20-specific CD8+ T cells in naïve and immunized mice

The in vivo activity of CUE-101 was tested in HLA-A2 transgenic mice that express a chimeric HLA class I molecule composed of the α1 and α2 domains from human HLA-A2.1 and the α3 transmembrane/cytoplasmic domain from murine H-2Dd (26). Weekly intravenous treatment of immunologically naïve HLA-A2 mice with CUE-101 elicited a population of E711-20-specific CD8+ T cells in the blood and spleen that was not observed with vehicle treatment (Fig. 3A and B; Supplementary Fig. S3). No significant changes in the frequencies of CD4+, CD8+, Treg, NKT, or NK nontarget cells were observed in the peripheral blood or spleen, demonstrating antigen-specific cellular targeting with CUE-101 in vivo (Fig. 3C). CUE-101 was well tolerated with no observations of treatment-related clinical signs or body weight loss. Effector T-cell activation, as characterized by antigen-specific IFNγ production following ex vivo peptide stimulation, confirmed that the antigen-specific T cells expanded through in vivo treatment possess CTL-like properties (Fig. 3D and E).

The ability of CUE-101 to expand an existing population of antigen-specific CD8+ memory T cells was assessed in HLA-A2 transgenic mice that were first immunized with E711-20 peptide to generate a specific CD8+ T-cell population and then randomized on the basis of the frequency of E711-20-specific CD8+ T cells. Mice were rested for 3 weeks after the final boost (baseline; Fig. 3F) and then treated with vehicle or CUE-101. As in the naïve setting, intravenous treatment with CUE-101 resulted in expansion of the E711-20-specific CD8+ T-cell population in blood relative to the baseline frequency prior to treatment, whereas vehicle treatment did not result in E7-specific T-cell expansion (Fig. 3G). The functionality of the expanded CD8+ T cells was demonstrated via detection of antigen-specific IFNγ production following ex vivo peptide stimulation (Fig. 3H). Thus, intravenous treatment with CUE-101 alone was sufficient to expand an E711-20-specific population from both a naïve CD8+ T-cell repertoire and an existing T-cell population.

Treatment with a murine surrogate of CUE-101 expands polyfunctional E749-57-specific CD8+ T cells in the tumor, spleen, and blood of TC-1 tumor-bearing mice

A murine surrogate of CUE-101 (mCUE-101) with functionally equivalent domains was generated to assess activity in immunocompetent tumor-bearing mice. mCUE-101 retains the design of CUE-101, with an effector attenuated murine IgG2a Fc, an H-2Db-E749-57 peptide–MHC complex, and identical human mutant IL2 components. Human IL2 is cross-reactive with mouse IL2R, modeling human IL2 effects on mouse cells and in mouse tumor models (27). As T-cell activation induces upregulation of PD-1 (PDCD1), and PD-1–blocking antibodies are approved for the treatment of patients with HNSCC, combination therapy with mCUE-101 and anti-PD-1 checkpoint blockade was also explored.

The TC-1 murine tumor model expresses the HPV16 E6 and E7 oncoproteins, and HPV16 E7 vaccination strategies were previously shown to mediate antitumor efficacy in this model (28) whereas single-agent anti-PD-1 treatment did not (29, 30). The antitumor activity of mCUE-101 was assessed in immunocompetent C57BL/6 mice with treatment initiation after TC-1 tumors were established. Significant tumor growth inhibition and prolongation of survival were observed after mCUE-101 administration, but no animals demonstrated complete tumor regression (Fig. 4A; Supplementary Fig. S4A). To assess the pharmacodynamic effects of treatment in this setting, tumors, spleens, and peripheral blood were harvested 6 days after the final treatment with mCUE-101 at a time when tumor growth inhibition was observed. Single-agent mCUE-101 was well tolerated, with only small, but significant, reductions in NK cells in the blood and CD4+ T cells in the spleen (Supplementary Fig. S4B). In the tumors, though, combination treatment was associated with a significant increase in the frequency of total CD8+ and CD4+ T cells as well as NK cells, suggesting enhanced tumor immune infiltration (Supplementary Fig. S4B). Furthermore, the frequency of E749-57-specific T cells among all tumor-infiltrating CD8+ T cells was significantly increased by single-agent mCUE-101 treatment (P < 0.001), and further enhanced by combination treatment, where on average >50% of the CD8+ tumor infiltrate was antigen-specific and there was also a significant increase in the CD8:Treg ratio (P < 0.001; Fig. 4BD). E7-specific CD8+ T cells were not expanded in tumors from mice treated with anti-PD-1 alone. In the spleens and peripheral blood of mCUE-101–treated animals, there was also a trend toward increased frequencies of E7-specific CD8+ T cells, which was significantly increased upon combination treatment (P < 0.01; Fig. 4E; Supplementary Fig. S4C). Responsiveness of the expanded T cells to cognate peptide presentation was assessed via intracellular cytokine staining of whole tumor-infiltrating lymphocyte (TIL) and splenocyte populations from treated animals that were stimulated ex vivo with E749-57 peptide. Among tumor-infiltrating CD8+ T cells, and consistent with the tetramer staining results, single-agent mCUE-101 treatment resulted in a significant increase in the frequency of cells that were positive for IFNγ and double positive for CD107a and Granzyme B (Gzmb) in response to peptide challenge (P < 0.01), and these frequencies were further increased upon combination treatment (P < 0.05; Fig. 4F). Similarly, increased frequencies of polyfunctional E7-specific CD8+ T cells were also observed in splenocytes (Fig. 4G), confirming that the E7-specific CD8+ cells expanded by mCUE-101 treatment were able to function as cytotoxic T lymphocytes and promote enhanced antitumor immunity. Significant upregulation of PD-1 surface expression on E7-specific T cells in mCUE-101–treated animals was observed when compared to the non-antigen–specific CD8+ T cells in the blood and spleen, and the magnitude of this effect was even greater within the tumor (P < 0.001; Fig. 4H), providing further confirmation of in situ activation of the tumor antigen-specific T cells expanded by mCUE-101 treatment.

Treatment with mCUE-101 promotes antitumor immunity and functional immunologic memory

The antitumor activity of mCUE-101 was further assessed by initiating treatment 24 hours after subcutaneous implantation of TC-1 tumor cells. Treatment with mCUE-101 significantly increased survival in this model (P = 0.005), expanded a population of E7-specific CD8+ T cells in the periphery, and resulted in 10% (1/10) of animals being tumor-free 79 days post-implantation (Fig. 5A and B; Supplementary Fig. S5A). This antitumor activity is specific to mCUE-101 as an analog targeting a different peptide specificity (LCMV GP3333-41; CUE-1843-835) did not inhibit tumor growth or expand E749-57-specific CD8+ T cells in this model, though it did expand a population of GP3333-41-specific CD8+ T cells (Supplementary Fig. S5B and S5C).

Combination treatment with mCUE-101 and anti-PD-1 further increased survival (P = 0.003), E7-specific CD8+ T-cell frequency (P < 0.05), and antitumor activity relative to mCUE-101 monotherapy, including an increase in the number of animals remaining tumor-free (60%; 6/10) up to 79 days postimplantation (Fig. 5A and B; Supplementary Fig. S5A). All treatments were well tolerated without clinical signs or body weight loss. Comparable increases in the frequency of E7-specific CD8+ T cells were observed in the tumors, spleens, tumor-draining lymph nodes, and peripheral blood of animals in the setting of treatment soon after tumor implantation (Supplementary Fig. S6A). Significant upregulation of PD-1 on tumor-infiltrating E7-specific CD8+ T cells was also observed upon treatment in this setting (P < 0.01; Supplementary Figs. S6B and S6C). This upregulation of PD-1 expression provides a mechanistic rationale underlying the significantly enhanced antitumor efficacy observed upon combination treatment with anti-PD-1 blockade, and demonstrates a consistency of effect in both early and established treatment settings that suggests the greater tumor control in the early treatment setting may be due to the earlier expansion of the relevant T-cell population prior to the rapid outgrowth of the tumor.

Induction of immunologic memory was assessed in mice that remained tumor-free 95 days after initial challenge and combination therapy by rechallenging animals with TC-1 tumors. Upon implantation all treatment naïve animals formed tumors, whereas animals previously treated with combination therapy rapidly rejected the tumors, demonstrating immunologic memory was established with mCUE-101 treatment (Fig. 5C). Depletion of CD8+ T cells in tumor-free mice prior to rechallenge resulted in tumor formation similar to that observed in tumor naïve mice, supporting the mechanism of action of mCUE-101 (Fig. 5D). In contrast, depletion of CD4+ T cells or treatment with an anti-IgG2b isotype control antibody was associated with significantly longer survival, suggesting that CD4+ T cells are not required for protective immunologic memory in this model. Together, these results demonstrate that a murine surrogate of CUE-101 is able to activate specific antitumor immunity, expand a therapeutically relevant frequency of E7-specific CD8+ T cells, and induce functional immunologic memory, which is significantly enhanced in the setting of combination therapy with anti-PD-1 blockade. These results support the potential for CUE-101 to elicit and expand E7-specific CD8+ T cells to stimulate antitumor immunity in patients with HPV16-driven tumors, and provide a rationale to further test combination therapy with immune checkpoint blockade in these patients.

This report describes the application of the Immuno-STAT platform to the development of a novel therapeutic candidate, CUE-101. In model systems, CUE-101 demonstrates selective activation and expansion of antigen-specific CD8+ T cells that mediate tumor clearance and establish antitumor immunologic memory. These preclinical data and the novel mechanism of action support the use of CUE-101 to address multiple HPV16-driven malignancies. CUE-101 is currently being tested as a monotherapy in HPV16+ patients with HNSCC (NCT03978689).

Clinical relevance of tumor-specific T cells for cancer immunotherapy is demonstrated by objective clinical responses after adoptive transfer of autologous TILs, including when only a few tumor-targeting T-cell specificities were represented in the TIL population (31, 32), or when T cells were engineered to express a single TCR (33). However, these therapies require extensive and specialized ex vivo manipulation of patient-derived cells and challenging preconditioning regimens to promote survival of the transferred cells. Recombinant IL2 (aldesleukin) therapy also enhances antitumor immunity (34), but toxicities associated with aldesleukin curbs its use in the clinic and expansion of Tregs may limit its effectiveness (35, 36). Vaccination approaches can have similar limitations, where long peptides have generated both effector and Treg responses (37). In contrast, CUE-101 is a single molecule with the minimal essential signals for direct activation of antigen-specific CD8+ T cells, functioning as an off-the-shelf therapeutic to enhance antigen-specific T-cell responses without requiring ex vivo manipulation. Functional attenuation of the IL2 components of CUE-101 reduces the potential for aldesleukin-associated toxicities, and for activation of other IL2R expressing cell types that may limit its activity. As a result, CUE-101 exposure leads to selective expansion of E711-20-specific CD8+ T cells without significant expansion of Tregs in vitro and in vivo, and its peptide-HLA specificity precludes the generation of an antigen-specific Treg response that could limit therapeutic potential.

CUE-101 targets a single peptide specificity, yet treatment of human PBMCs demonstrates that CUE-101 can activate and expand a polyclonal CD8+ T-cell repertoire, including multiple unique TCRs capable of recognizing and responding to the E711-20 peptide. Thus, in this molecular framework a single peptide–HLA complex does not limit response to a single TCR. Although E711-20 has been identified as an immunodominant HLA-A*0201 epitope (10), the E711-19 peptide is also naturally processed and presented on cancer cells (11). Thus, it is significant that the two TCRs identified and tested from CUE-101 expanded CD8+ T cells were able to recognize both the E711-20 and E711-19 peptides, suggesting that if only one of these peptides is processed and presented in a given patient CUE-101 will expand a relevant CTL population. Therefore, CUE-101 has the potential to harness a diverse, native T-cell repertoire to mount an effective antitumor immune response while avoiding systemic activation of the immune system.

The activity of a murine surrogate in the immunocompetent TC-1 tumor model provides a mechanistic rationale for combination activity of CUE-101 with PD-1 checkpoint blockade in the clinic. Activation of T cells leads to upregulation of PD-1 expression (38), which is consistent with the observed PD-1 upregulation on the surface of E7-specific CD8+ T cells after treatment with mCUE-101. CUE-101 treatment may thus lead to significant expansion of E711-20-specific CD8+ T cells in patients, but activation-induced expression of PD-1 may limit their ability to kill E7 expressing tumor cells. Preclinical data presented here support that CUE-101 in combination with a PD-1 blocking antibody may overcome this mechanism that might limit clinical activity. A recent clinical trial testing PD-1 blockade in combination with an E6/E7 vaccination approach resulted in a favorable clinical response rate (39), further supporting this combination approach with CUE-101. The therapeutic benefit of CUE-101 plus PD-1 blockade was not as strong in the late treatment setting despite achieving the desired pharmacodynamic effect in vivo, suggesting additional immune stimulation may be needed to treat advanced disease or to overcome an immunosuppressive tumor microenvironment. Further characterization of the tumor microenvironment and understanding mechanisms of escape in the TC-1 model may provide clinically relevant information for the development of CUE-101. For example, much work is ongoing in the field to develop agents specifically designed to overcome mechanisms of escape, which may represent opportunities to enhance antitumor immunity in combination with CUE-101. In addition, deep phenotypic characterization of the E711-20-specific CD8+ T cells expanded by CUE-101 preclinically and in patients may reveal additional rational combination approaches for future testing to overcome these potential limitations.

Although CUE-101 specifically targets E711-20 specific T cells, there is also potential for this class of molecules to have activity across different cancer indications. In support of this, the CMV- and LCMV-targeted analogs used in this study were also able to selectively activate and expand their respective T-cell specificities. Additional analogs targeting tumor-associated antigens represent potential candidates for development of molecules for other cancer indications. Ongoing and future research may also identify features of the tumor microenvironment where the optimal biological effect would be provided by an immunomodulatory signal other than IL2. The unique modular nature of this class of molecules thus provides flexibility with potential for broad applicability in the development of additional novel cancer treatments.

S.N. Quayle, D.R. Thapa, Z. Merazga, M.M. Kemp, A. Histed, F. Zhao, M. Moreta, P. Ruthardt, L. Witt, J. Ryabin, J.F. Ross, P.A. Kiener, R. Chaparro, R. Seidel, A. Suri, S. Cemerski, K.J. Pienta, and M.E. Simcox are employees/paid consultants for and hold ownership interest (including patents) in Cue Biopharma. N. Girgis holds ownership interest (including patents) in Cue Biopharma. D.R. Beal, J. Soriano, M. Haydock, and E. Spaulding are employees/paid consultants for Cue Biopharma. S. Almo is an employee/paid consultant for, reports receiving commercial research grants from, and holds ownership interest (including patents) in Cue Biopharma. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S.N. Quayle, N. Girgis, Z. Merazga, S. Hulot, A. Nelson, J.F. Ross, P.A. Kiener, S. Almo, R. Chaparro, R. Seidel, A. Suri, S. Cemerski, K.J. Pienta, M.E. Simcox

Development of methodology: N. Girgis, D.R. Thapa, Z. Merazga, S. Hulot, A. Nelson, D.R. Beal, E. Spaulding, S. Almo, K.J. Pienta

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Girgis, D.R. Thapa, Z. Merazga, M.M. Kemp, A. Histed, F. Zhao, M. Moreta, P. Ruthardt, S. Hulot, A. Nelson, L.D. Kraemer, L. Witt, J. Ryabin, J. Soriano, M. Haydock, E. Spaulding, S. Cemerski

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.N. Quayle, N. Girgis, D.R. Thapa, Z. Merazga, M.M. Kemp, A. Histed, F. Zhao, M. Moreta, P. Ruthardt, S. Hulot, A. Nelson, L.D. Kraemer, D.R. Beal, L. Witt, J. Ryabin, J. Soriano, E. Spaulding, P.A. Kiener, S. Cemerski, K.J. Pienta

Writing, review, and/or revision of the manuscript: S.N. Quayle, N. Girgis, D.R. Thapa, Z. Merazga, M.M. Kemp, A. Histed, F. Zhao, M. Moreta, P. Ruthardt, S. Hulot, D.R. Beal, J.F. Ross, P.A. Kiener, S. Almo, R. Chaparro, A. Suri, S. Cemerski, K.J. Pienta, M.E. Simcox

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.N. Quayle, N. Girgis, Z. Merazga, M. Moreta, S. Hulot, L.D. Kraemer, L. Witt, J. Soriano, S. Cemerski

Study supervision: S.N. Quayle, N. Girgis, M. Moreta, J.F. Ross, P.A. Kiener, A. Suri, S. Cemerski

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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