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CUE-101, a Novel Fc Fusion Protein for Selective Targeting and Expansion of Anti-Tumor T Cells for Treatment of HPV-Driven Malignancies

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CUE-101, a Novel Fc Fusion Protein for Selective Targeting and Expansion of Anti-Tumor T Cells for Treatment of HPV-Driven Malignancies CUE-101, a novel Fc fusion protein for selective targeting and expansion of anti-tumor T cells for treatment of HPV-driven malignancies Natasha Girgis1, Steven N. Quayle1, Alyssa Nelson1, Dharma Raj Thapa1, Sandrine Hulot1, Lauren Kraemer1, Miguel Moreta1, Zohra Merazga1, Robert Ruidera1, Dominic Beal1, Mark Haydock1, Jonathan Soriano1, Luke Witt1, Jessica Ryabin1, Emily Spaulding1, John F. Ross1, Saso Cemerski1, Anish Suri1, Rodolfo Chaparro1, Ronald Seidel1, Kenneth J. Pienta2, Mary C. Simcox1 1Cue Biopharma, Cambridge, Massachusetts; 2The James Buchanan Brady Urological Institute and the Department of Urology, Johns Hopkins School of Medicine, Baltimore, Maryland + + Background CUE-101 selectively expands E711-20-specific CD8 T mCUE-101 expands functional antigen-specific CD8 T • Human papilloma virus (HPV) is responsible for 72% of oropharyngeal, 90% of cervical, 90% cells from healthy human PBMCs cells in the tumor and the periphery of anal, and 71% of vulvar, vaginal, or penile cancers, causing significant morbidity and A. B. A. B. C. 9 mortality worldwide. Innovative therapies are urgently needed for these malignancies, Vehicle 100 nM CUE-101 107 15 Peripheral Blood particularly in the largely incurable metastatic setting. 106 10 5 • The E7 oncoprotein is constitutively expressed in HPV-associated cancers, is necessary for 105 ⍺PD-1 5 PE 4 Vehicle mCUE-101 Combo - 10 initiation and maintenance of malignant transformation, and is genetically conserved in 1 cancer (Mirabello 2017). 103 1 1 102 E7-Specific of CD8+ T cells % IFNg+,TNFa+ • Clinical proof of concept for HPV-targeted T cell therapy includes demonstration of complete Tetramer CD8+ T Cells 1 20 (absolute counts) regression of metastatic cervical cancer upon adoptive transfer of tumor-infiltrating T cells - 10 11 Immune Cell Subsets E7 Tetramer E7 100 AgS among CD8+ T cells % (Stevanovic 2015; Stevanovic 2017) E7 0.01 0.1 1 10 100 1000 10000 0 0 E711-20 Tetramer-APC [CUE-101] (nM) E7 Tetramer • The E7 sequence, including that encoding the E711-20 peptide in CUE-101, is maintained in PD1 E7 -specific CD8+ T cells CD4+ T cells α PD-1 11-20 Vehicle Combo α cancer and this epitope is immunodominant in humans (Ressing 1995). CD8+ T cells CD4+ CD25+ Foxp3+ T cells Vehicle Combo • Immuno-STATTM molecules are engineered to selectively modulate the activity of antigen- NK cells mCUE-101 mCUE-101 + specific T cells in situ CUE-101 selectively expands E711-20-specific CD8 T cells from whole human PBMCs in vitro. (A) D. E. F. Primary human PBMCs were exposed to increasing concentrations of CUE-101 for 10 days. CUE-101 100 15 + Tumor elicited a population of E711-20-specific CD8 T cells measured by tetramer staining, while vehicle treatment CUE-101 did not. (B) Expansion of E7-specific CD8+ T cells occurred in a dose-dependent manner. Increasing 75 Vehicle mCUE-101 ⍺PD-1 Combo 10 expansion of total NK and total CD8+ cells was also observed in response to CUE-101 treatment. A. B. 50 5 of CD8+ T cells pHLA CUE-101 selectively expands functional HPV E711-20- 25 % IFNg+,TNFa+ + AgS among CD8+ T cells % E7 Tetramer E7 specific CD8 T cells in HLA-A2 transgenic mice 0 0 T Cell E7 Tetramer IL-2 Naïve HLA-A2 transgenic mice PD1 α PD-1 2 20 α T Cell Vehicle Vehicle Combo Vehicle Combo T Cell T Cell mCUE-101 A. 30 mg/kg B. 1 C. Peptide mCUE-101 Vehicle CUE-101 0.5 30 mg/kg T Cell 10 Fc T Cell CUE-101 Mice bearing established TC-1 tumors were treated with 15 mg/kg mCUE-101 alone or in combination with 0.4 αPD-1. Expansion of Ag-specific cells was assessed one week after the last dose of mCUE-101. + 0.3 3 Representative flow plots show the frequency of tetramer-positive CD8 T cells in the blood (A & B) and tumor (D & E). Only animals treated with mCUE-101 exhibited increased frequency of Ag-specific T cells, 0.2 2 + % of Total Cells % of Total Tetramer which was greatly increased in the tumor vs blood. mCUE-101 increased the frequency of CD8 T cells Schematic of CUE-101 design and mechanism of action. (A) CUE-101, a novel human fusion protein, 0.1 1 that produced IFNɣ and TNF⍺ in response to E749-57 peptide restimulation of splenocytes (C) and tumor- is comprised of a human leukocyte antigen (pHLA) complex, HLA-A*0201, with a peptide epitope derived AgS among CD8+ T cells % infiltrating lymphocytes (F). from the HPV16 E7 protein (amino acid residues 11-20), a reduced affinity human interleukin-2 (IL-2) 0.0 0 Tetramer Vehicle Peptide 30 mg/kg variant, and an effector attenuated human immunoglobulin G (IgG1) Fc domain. (B) CUE-101 is proposed NK CUE-101 NKT to selectively bind and activate antigen-specific CD8+ T cells endogenously present in patients with CD4+ CD8+ Treg Surrogate mCUE-101 upregulates PD-1 expression on HPV16-driven malignancies. Upon binding and activation, target CD8+ T cells are stimulated to proliferate Peptide-immunized HLA-A2 transgenic mice and eradicate the tumor. + D. Baseline Day 35 E. F. tumor-infiltrating E7-specific CD8 T cells 20 90 Vehicle 10000 60 mCUE-101 AgS CD8+ Vehicle Peptide A. B. *** 30 Blood Tumor Non AgS CD8+ CUE-101 selectively binds antigen-specific T cells and 25 10 30 mg/kg 8000 *** CUE-101 20 elicits effector cytokine production 6000 3 15 Mode 4000 + Tetramer 10 2 gMFI of PD1 CUE-101 selectively binds antigen (Ag)-specific CD8 T cells Cells % of Total 1 % AgS among CD8 T Cells % 5 2000 30 mg/kg *** 0 0 PD-1 A. B. C. CUE-101 Non-AgS CD8+ T cells + + Vehicle Peptide 30 mg/kg 0 Ag-specific CD8 T Cells Primary CD8 T Cells Primary NK Cells NK CUE-101 NKT AgS CD8+ T cells Blood Tumor CD4+ CD8+ Treg 70 90 90 HPV E711-20 Specific CD8+ T Cells HPV E711-20 Specific CD8+ T Cells HPV E711-20 Specific CD8+ T Cells Isotype control CMV pp65 Specific CD8+ T Cells 80 Naive CD8+ T Cells (Donor #1) NK cells (Donor #1) Tetramer 60 495-503 80 Naive CD8+ T Cells (Donor #2) NK cells (Donor #2) 70 70 NK cells (Donor #3) + + + 50 Naive CD8+ T Cells (Donor #3) CUE-101 expands E711-20-specific CD8 T cells in HLA-A2 mice. (A-C) Naïve HLA-A2 transgenic mice (A) Flow histograms display PD-1 expression on E7-specific CD8 T cells (black) vs non-E7-specific CD8 60 60 were dosed intravenously (IV) once weekly for 5 weeks with CUE-101 and the frequency of E7 - T cells (gray), or isotype control (dashed line) in peripheral blood and tumor. (B) mCUE-101 treatment 40 50 50 11-20 + + 30 40 40 specific CD8 T cells was assessed in peripheral blood. Ag-specific CD8+ T cells expanded in response to significantly increased (p<0.001) PD-1 expression levels (gMFI) on E7-specific CD8 T cells present within 30 30 20 CUE-101 treatment (A & B) without broadly affecting other immune lineages (C). (D-F) HLA-A2 the TC-1 tumors, providing rationale for combo therapy. 20 20 % CUE-101 bound cells % CUE-101 bound cells % CUE-101 bound cells 10 transgenic mice were immunized with E711-20 peptide and rested to allow Ag-specific T cells to contract 10 10 prior to IV dosing of CUE-101 on Days 1, 8, and 29. Preexisting Ag-specific CD8+ T cells expanded in 0 0 0 + 0 0.01 0.1 1 10 100 1000 0 0.01 0.1 1 10 100 1000 0 0.01 0.1 1 10 100 1000 response to CUE-101 treatment (D & E) and repeated CUE-101 treatment did not broadly affect the Treatment with mCUE-101 results in functional CD8 T [CUE-101] (nM) [CUE-101] (nM) [CUE-101] (nM) peripheral immunophenotype (F). Naïve Immunized cell-dependent immunological memory CUE-101 potently and selectively binds to E7-specific T cells but not to CMV pp65495-503-specific CD8+ T A. 30 mg/kg B. C. + HLA-A2 HLA-A2 cells (A), primary naïve CD8 T cells (B), or primary NK cells (C) that also express IL-2 receptor (IL-2R). Vehicle CUE-101 0.4 100 A. 1500 B. 100 0.3 Isotype 50 ) CD8 depleted CUE-101 selectively induces signaling and effector 3 1200 Tumor Naive + 75 cytokine production in antigen-specific CD8 T cells 0.2 20 CD45 900 ** + of CD8+ T cells γ 50 0.1 10 600 % IFNy+ of CD8+ T cells % IFN- A. E7-specific CD8+ T Cells B. CMV-specific CD8+ T Cells Naive 120 CUE-101 120 CUE-101 IFN-ɣ 0.0 0 300 25 Tumor volume (mm Combo Treated Overall Survival (%) CMV Immuno-STAT CMV Immuno-STAT Vehicle 30 mg/kg Vehicle 30 mg/kg CUE-101 CUE-101 100 Naive CD8 T cells 100 Naive CD8 T cells ns P u rifie d C M V -s p e c ific C D 8 + T c e lls 0 ) + 0 g Cytokine production in E7 -specific CD8 T cells expanded by CUE-101. Treatment of naïve (A & 80 80 11-20 0 20 40 60 N 2 0 0 10 20 30 40 50 F I ( C M V Im m u n o S T A T B) or peptide -immunized (C) HLA-A2 transgenic mice with CUE-101 as described above increases Days Post Rechallenge Days Post Engraftment s l 60 60 l 1 5 e frequencies of CD8+ T cells in the spleen that produce IFN-ɣ in response to E711-20 peptide, demonstrating C (A) Mice remaining tumor-free after combination treatment were rechallenged with TC-1 tumors 97 days g 40 40 that CUE-101 expands functional CD8+ T cells in vivo.
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