RTX-321, an Allogeneic Artificial Antigen Presenting Cell (aAPC) Red Cell Therapeutic, Expressing MHC I-Peptide, 4-1BBL and IL-12, Promotes Expansion and Anti-Tumor Activity of Antigen-Specific T Cells in HPV16-Positive Tumors Xuqing Zhang, Mengyao Luo, Shamael R. Dastagir, Mellissa Nixon, Albert Lee, Naren Subbiah, Douglas C. McLaughlin, Chris Moore, Sneha Pawar, Nicholas Bayhi, Timothy J. Lyford, Omkar Bhate, Viral Amin, Christopher L. Carpenter, Thomas J. Wickham, and Tiffany F. Chen Rubius Therapeutics, Cambridge, MA, USA Poster #P233 Figure 10. RTX-321 (HPV-4-1BBL-IL-12) is Active in HPV-Specific TCR Transduced T Cell Line and Primary Human T Cells (A) RTX-321 expression was determined by anti-IL-12, anti-4-1BBL and anti-β2M staining. (B) 4×10 5 Jurkat cells with a NFAT luciferase reporter engineered to express HPV E7-specific TCR were incubated with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12 or RTX-321 at a range of doses (3.2×10 6 , 1.6×10 6 , 8×10 5 , 4×10 5 , 2×10 5 , 1×10 5 ). After 18- to 22-hour incubation, a luciferase reporter assay was carried out to determine NFAT signal-fold change compared to Jurkat reporter cell alone. (C) 3.5×10 4 4-1BB/NFB reporter HEK293 cell line that expresses human 4-1BB and has a NFκB luciferase reporter construct stably integrated was incubated at a range of doses of RTX-321 and controls (4×10 5 , 2×10 5 , 1×10 5 , 5×10 4 , 2.5×10 4 , 1.25×10 4 , or 6.25×10 3 ). After 18 to 22-hour incubation, a luciferase reporter assay was carried out to determine NFκB signal fold-change compared to reporter cell alone. (D) 5×10 4 HEK-Blue IL-12 cells, expressing a STAT4-inducible SEAP reporter gene and the IL-12 receptors, were incubated with RTX-321 and controls at different doses (5×10 5 , 1.7×10 5 , 5.6×10 4 , 1.9×10 4 , 6.2×10 3 , or 2.1×10 3 ) for 24 hours. SEAP expression was measured by absorbance at 655 nm and shown as fold-change relative to the reporter cell alone control. (E) CD8+ T cells from a human donor were transduced with lentivirus to express an HPV E7-specific TCR. 8×10 4 TCR engineered primary CD8+ T cells with ~31% TCR expression, and untransduced T cells were incubated with 1.6×10 5 RTX-321 and controls for 7 days. HPV E7 tetramer+ CD8 T cell numbers were determined by flow cytometry. 4-1BBL = 4-1BB ligand; β2m = beta-2-macroglobulin; CTRL = control; HPV = HLAA2-human papilloma virus peptide; IL = interleukin; NFAT = nuclear factor of activated T cells; NFκB = nuclear factor kappa-light-chain-enhancer of activated B cells; RCT = Red Cell Therapeutic experimental construct; RTX = Red Cell Therapeutic product candidate; RTX-321 = red cell therapeutic-HPV-4-1BBL-IL-12; SEAP = Secreted embryonic alkaline phosphatase; SSC = side scatter; TCR = T cell receptor; UNT = untransduced control; HLAA2 = human leukocyte antigen A2. • RTX-321 selectively directs against HPV dominant epitope in HPV+ cancer patients and robustly engages TCR, 4-1BB and IL- 12R signaling in engineered cell lines • RTX-321 expands HPV-specific TCR-transduced primary human T cells CONCLUSIONS • RTX-aAPCs activate and significantly expand antigen-specific T cells, reduce tumor burden and have minimal, reversible toxicity • Expression of signal 3 on an RTX-aAPC results in robust antigen-specific T cell expansion, memory formation and cytotoxicity towards tumor cells • RTX-aAPC targeted against a tumor-associated antigen, gp100, promotes antigen-specific T cell expansion and effector function, and nearly eliminates B16-F10 lung metastases at the highest doses • RTX-aAPC targeting CMV epitope expands CMV-specific T cells from CMV+ healthy donor PBMC • RTX-321 (HPV-4-1BBL-IL-12) activates an HPV-specific TCR transduced T cell line and primary human T cells • Clinical studies are planned to evaluate RTX-321 for the treatment of patients with HPV 16+ cancers, including cervical and head and neck cancer ACKNOWLEDGEMENTS Poster design support was provided by Dennig Marketing Group, sponsored by Rubius Therapeutics. We would like to thank Lori Melançon for her editorial contributions. DISCLOSURES All authors: Employment with and equity ownership in Rubius Therapeutics. RESULTS AND METHODS Figure 5. IL-12 as Signal 3 on aAPC Significantly Enhances Efficacy and Potency in a Subcutaneous EG7.OVA Tumor Model (A) CD45.1 Pep Boy mice were injected subcutaneously with 2×10 6 EG7.OVA cells. When the tumors reached a volume of approximately 150 mm 3 , the animals were randomized and dosed on Day 1 post randomization with 1×10 6 naïve OT1 cells. The animals were then dosed on Days 1, 4 and 7 with 2.5×10 8 of mRBC-CTRL, mRBC-OVA-4-1BBL, mRBC-OVA-4-1BBL-IL-7, mRBC-OVA-4-1BBL-IL-12 or mRBC-OVA-4-1BBL-IL-15. An additional control group received 200 μL PBS. (B) Tumor volumes were monitored every 2-3 days. (C) Survival and (D) tumor volume and regression were monitored in 1×10 9 mRBC-CTRL or mRBC-OVA-4-1BBL-dosed mice as well as 4-fold lower (2.5×10 8 ) and 16-fold lower (6.25×10 7 ) mRBC-OVA-4-1BBL-IL-12-dosed mice. 4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; IL = interleukin; mRBC = murine red blood cell; OVA = H2K b -ovalbumin; PBS = phosphate buffered saline. • mRBC-OVA-4-1BBL-IL-12 demonstrates superior tumor control efficacy compared to mRBC-OVA-4-1BBL, mRBC-OVA-4- 1BBL-IL-7 or mRBC-OVA-4-1BBL-IL-15. Based on these results, IL-12 is chosen as signal 3 for RTX-aAPC • mRBC-OVA-4-1BBL-IL-12 significantly increases survival • A significant number of tumor regressions were observed at the 2.5×10 8 and 6.25×10 7 doses of mRBC-OVA-4-1BBL-IL-12 Figure 6. mRBC-OVA-4-1BBL-IL-12 Treatment Results in Minimal, Reversible Toxicity In Vivo, Likely Due to Restriction to the Vasculature (A) Wildtype mice were dosed with 1×10 9 mRBC-CTRL or 1×10 9 , 3×10 8 , or 6×10 7 mRBC-OVA-4-1BBL-IL-12 on Days 0, 4, 7 and 11. Mice were sacrificed on Day 12 or on Day 25. (B) Body weight changes compared to Day 0 were monitored overtime. (C) IFNγ level in plasm was determined by cytokine bead array overtime. (D) Serum alanine aminotransferase (ALT) levels on Days 12 and 25 were determined by clinical chemistry. 4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; ALT = alanine aminotransferase; CTRL = control; IFNγ = interferon γ; IL = interleukin; mRBC = murine red blood cell; OVA = H2K b -ovalbumin. • No significant body weight changes were observed • Serum IFNγ level returned to baseline after a recovery period • Elevated serum alanine aminotransferase returned to baseline after a recovery period Figure 7. mRBC-OVA-4-1BBL-IL-12 Expands Endogenous OVA-Specific T cells CD45.1 Pep Boy mice were transferred with 2×10 6 naïve OT1 cells on Day 0 followed by dosing with 1×10 9 mRBC-CTRL or 1×10 9 , 3×10 8 , or 6×10 7 mRBC- OVA-4-1BBL-IL-12 on Days 0, 4, 7 and 11. Mice were sacrificed on Day 12 to determine OT1 and OVA-tetramer+ T cell number in (B) 30 μL blood and (C) spleen. 4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; IL = interleukin; mRBC = murine red blood cell; OVA = H2K b -ovalbumin. • mRBC-OVA-4-1BBL-IL-12 drives expansion of endogenous OVA-specific T cells. The expansion of the endogenous OVA- specific T cells is comparable to that of the OT1 cells one day after last dose in circulation and the spleen Figure 3. The Addition of IL-7, IL-12 or IL-15 as Signal 3 on an RTX-aAPC Further Promotes Antigen-Specific T cell Expansion, Memory Formation and Cytotoxicity Towards Tumor Cells 1×10 5 OT1 T cells were incubated with 1×10 6 , 3.3×10 5 or 1×10 5 mRBC-CTRL, mRBC-OVA-4-1BBL, mRBC-OVA-4-1BBL-IL-7, mRBC-OVA-4-1BBL-IL-12 or mRBC-OVA-4-1BBL-IL-15 at 37°C for 4 days. (A) OT1 CD8+ T cells were cultured with RCT-OVA-4-1BBL, RCT-OVA-4-1BBL-IL-7, RCT-OVA-4-1BBL-IL-12 or RCT-OVA-4-1BBL-IL-15 at 1:10 ratio. OT1 cells were harvested after a 3-day incubation and treated with ACK buffer to lyse the RTX-aAPC. 1×10 4 tumor cells, either parental tumor cells, EL4, or tumor cells expressing ovalbumin, EG7.OVA, were then incubated with expanded OT1 cells (effector cells) at 5:1, 2:1, 1:1, 0.5:1 and 0:1 effector to target (E:T) ratio. After a 22-hour incubation, cells were stained with live/dead dye and fixed with 2% paraformaldehyde to enumerate live target cells in each well. (B) OT1 numbers, (C) Tscm (CD122+CD62L+CD44-) OT1 number, (D) Tcm (CD122+CD62L+CD44+) OT1 number, and (E) Tem (CD122+CD62L - CD44+) OT1 number were quantified by flow cytometry. (F) EL4 percent killing and (G) EL7.OVA percent killing were calculated by live tumor cell percent of target only controls. 4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; IL = interleukin; MHC = major histocompatibility complex; mRBC = murine red blood cell; OVA = H2K b - ovalbumin; RCT = Red Cell Therapeutic experimental construct; RTX-aAPC = Red Cell Therapeutic artificial antigen presenting cell; Tcm = central memory T cell; Tem = effector memory T cell; TCR = T cell receptor; Tscm = stem cell memory T cell. • IL-7, IL-12 and IL-15 as signal 3 on a mouse (m) RBC-aAPC increases total antigen-specific T cell expansion and memory formation compared to mRBC-aAPC that expresses only signals 1 and 2 • IL-12 as signal 3 significantly improves antigen-specific killing of tumor cells compared to T cells expanded by RCT-OVA-4-1BBL, RCT-OVA-4-1BBL-IL-7 or RCT-OVA-4-1BBL-IL-15 Figure 4. IL-12 as Signal 3 on aAPC Promotes Antigen-Specific T Cell Expansion and Effector Function In Vivo (A) CD45.1 Pep Boy mice were inoculated subcutaneously with 2×10 6 EG7.OVA cells. When the tumors reached a volume of approximately 180 mm 3 , the animals were randomized and treated on Day 0 with 1×10 6 naïve OT1 cells. 1×10 9 mRBC-CTRL, mRBC-OVA-4-1BBL, mRBC-OVA-4-1BBL-IL-7, mRBC-OVA-4-1BBL-IL-12 or mRBC-OVA-4-1BBL-IL-15 was administered on Days 0 and 3. A control group received 200 μL of PBS. (B) Total OT1 number (CD45.2+CD8+) and (C) Memory OT1 number (Tscm:CD122+CD62L+CD44 - ; Tcm: CD122+CD62L+CD44+; Tem: CD122+CD62L - CD44+) in 50 μL blood on days 0, 3 and 6 were determined by flow cytometry. (D) Mice were sacrificed on Day 6 to evaluate OT1 number in tumor, draining lymph nodes and spleen. (E) Genomic DNA was extracted from single cell suspension of the tumor and OT1 clone frequency within tumor infiltrating T cells was determined by TCR sequencing. (F) Memory OT1 numbers in the spleen on Day 6 were determined by flow cytometry. (G) Splenocytes were stimulated with PMA and ionomycin and OT1 effector function was determined by intracellular cytokine staining. 4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; dLN = draining lymph node; GzmB = granzyme B; IFNγ = interferon γ; IL = interleukin; mRBC = murine red blood cell; OVA = H2K b -ovalbumin; Tcm = central memory T cell; Tem = effector memory T cell; Tscm = stem cell memory T cell. • mRBC-OVA-4-1BBL-IL-12 and mRBC-OVA-4-1BBL-IL-15 induce dramatic expansion of circulating OT1 in vivo compared to mRBC-OVA-4-1BBL or mRBC-OVA-4-1BBL-IL-7 • Circulating OT1 cells expanded by mRBC-OVA-4-1BBL-IL-12 and mRBC-OVA-4-1BBL-IL-15 have a predominant effector phenotype while maintaining a central memory T cell population • mRBC-OVA-4-1BBL-IL-12 induces >100-fold expansion of OT1 in the spleen compared to mRBC-CTRL • Significantly more OT1 T cells are detected in the tumor of mRBC-OVA-4-1BBL-IL-12-dosed mice compared to mRBC-CTRL by TCR sequencing • mRBC-OVA-4-1BBL-IL-12 maintained high level central memory T cell expansion in secondary lymphoid organs as seen with mRBC-OVA-4-1BBL • mRBC-OVA-4-1BBL-IL-12 promotes antigen-specific T cell functionality (IFNγ and Granzyme B) as well as increases polyfunctional (IFNγ+ and Granzyme B+) T cells INTRODUCTION • Red Cell Therapeutics™ (RCTs) are a new class of allogeneic, off-the-shelf cellular therapeutic candidates for the treatment of cancer, rare diseases and autoimmune diseases • For the treatment of cancer, allogeneic Red Cell Therapeutic artificial antigen presenting cells (RTX-aAPCs) are engineered to induce a tumor-specific immune response by expanding antigen-specific T cells. Rubius Therapeutics’ first artificial antigen-presenting cell product candidate, RTX-321, is for the potential treatment of HPV 16-positive cancers Figure 1. The RED PLATFORM ® is Designed to Generate Allogeneic, Off-the-Shelf Cellular Therapies MHC = major histocompatibility complex. • The enucleated reticulocytes are RCTs that express hundreds of thousands of biotherapeutic proteins on the cell surface • Delivered at a dose of <1% of total red blood cell volume in the body • Universal, scalable and consistent manufacturing process Figure 2. RTX-aAPC Drives Antigen-Specific Activation and Proliferation of T cells MHC = major histocompatibility complex; RTX-aAPC = Red Cell Therapeutic artificial antigen-presenting cell; TCR = T cell receptor. • RTX-aAPCs are engineered to simultaneously express on the cell surface a tumor- specific antigen on the MHC I, a co-stimulatory signal and a cytokine to mimic the human immunobiology of T cell-APC interactions OBJECTIVES • To determine the optimal signal 3 on an RTX-aAPC to promote robust antigen-specific T cell expansion, memory formation and cytotoxicity towards tumor cells • To determine whether RTX-aAPCs have broad antigen applicability, using OVA, gp100 peptide and HPV antigens Signal 3 cytokine Signal 2 co-stimulatory agonist Antigen-specific TCR MHC I Signal 1 Tumor antigen RTX-aAPC T CELLS Figure 8. aAPC Targeted Against a Tumor-Associated Antigen, gp100, Promotes Antigen-Specific Pmel-1 T Cell Expansion, Effector Function and Dramatically Reduces B16-F10 Lung Metastases (A) Wildtype mice were injected intravenously with 1×10 5 B16-F10 tumor cells on Day 0 followed by transfer of 2×10 6 naïve Pmel-1 T cells on Day 1. Mice were then dosed with 1×10 9 mRBC-CTRL or 1×10 9 , 2.5×10 8 , or 6×10 7 mRBC-gp100-4-1BBL-IL-12 on Days 1, 4, and 8. Mice were sacrificed on Day 14. (B) Representative lung photos of 1×10 9 mRBC-CTRL and 1×10 9 mRBC-gp100-4-1BBL-IL-12 dosed mice. (C) Lung metastasis counts in all groups were enumerated. Pmel-1 T cell number in (D) 50μL blood overtime, (E) the spleen, and (F) the left lobe of perfused lung were determined by flow cytometry. (G) Single-cell suspensions from perfused lung were stimulated with PMA and ionomycin followed by intracellular cytokine staining to determine effector function of lung infiltrating Pmel-1 and endogenous CD8+ T cells. 4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; GzmB = granzyme B; gp100, H2D b -gp100; IFNγ = interferon γ; IL = interleukin; mRBC = murine red blood cell. • mRBC-gp100-4-1BBL-IL-12 nearly eliminates lung metastases at the highest dose levels and dramatically reduces lung metastases at low dose levels in a B16-F10 melanoma model • mRBC-gp100-4-1BBL-IL-12 promotes antigen-specific Pmel-1 T cell expansion in circulation and secondary lymphoid organs • mRBC-gp100-4-1BBL-IL-12 increases lung-infiltrating antigen-specific Pmel-1 T cells and their effector function Figure 9. RCT-CMV-4-1BBL-IL-12 Expands CMV-Specific T cells From CMV+ Healthy Donor PBMC 2×10 5 HLAA2+CMV+ human PBMC were incubated with 8×10 5 RCT-CTRL, RCT-CMV, RCT-CMV-4-1BBL or RCT-CMV-4-1BBL-IL-12 at 37ºC for 10 days. (A) CMV tetramer+ CD8 T cell numbers and (B) their memory phenotype were determined by flow cytometry. (C) IFNγ producing CMV-specific T cells numbers were quantified by ELISPOT post CMV peptide stimulation with representative images in (D). 4-1BBL = 4-1BB ligand; CMV = HLAA2-cytomegalovirus peptide; CTRL = control; IFNγ = interferon γ; IL = interleukin; PBMC = periphery blood mononuclear cell; RCT = Red Cell Therapeutic experimental construct; Tcm = central memory T cell; Tem = effector memory T cell; Temra = terminally differentiated effector memory; Tscm = stem cell memory T cell; TNTC = too numerous to count. • RCT-CMV-4-1BBL-IL-12 expands CMV-specific T cells from healthy donor PBMC by both tetramer staining and ELISPOT measurements • RCT-CMV-4-1BBL-IL-12 promotes memory generation within CMV tetramer+ population Society for Immunotherapy of Cancer / Nov. 6-10, 2019 / National Harbor, ME RED PLATFORM ® EXPANSION & DIFFERENTIATION PROGENITOR CELL COLLECTION ONE HEALTHY O- DONOR ENUCLEATION & MATURATION GENETIC ENGINEERING MHC-PEPTIDE SIGNAL 2 AGONIST 100-1000’s OF DOSES RED CELL THERAPEUTIC 0 1 10 6 2 10 6 3 10 6 4 10 6 0 2 4 6 8 10 TCR Activity Cell Number Fold Change in Luminesence to HPV TCR+ Jurkat NFAT Reporter RCT-CTRL RCT-IL-12 RCT-HPV RCT-4-1BBL RTX-321 β2m 4-1BBL IL-12 SSC RTX-321 4-1BBL Activity 0 1 10 5 2 10 5 3 10 5 4 10 5 5 10 5 0 5 10 15 Cell Number Fold Change in Luminescence Over Hek-4-1BB-NF B Alone RCT-CTRL RCT-IL-12 RCT-HPV RTX-321 0 2 10 5 4 10 5 6 10 5 0 20 40 60 80 100 IL-12 Activity Cell Number Fold Change in SEAP to Media Alone CTRL RCT-CTRL RCT-IL-12 RCT-HPV RCT-4-1BBL RTX-321 RCT-CTRL RCT-HPV-4-1BBL RCT-4-1BBL-IL-12 RTX-321 0 200 400 2000 4000 6000 8000 10000 CD8 + HPV-E7 Tetramer + Number Total CD8 + HPV-E7 Tetramer + Count Day 7 UNT Day 7 HLAA2-HPV TCR A B C D E RCT-CTRL RCT-CMV RCT-CMV-4-1BBL RCT-CMV-4-1BBL-IL-12 0 5 10 15 20 Donor 1 CMV Tetramer + Counts RCT-CTRL RCT-CMV RCT-CMV-4-1BBL RCT-CMV-4-1BBL-IL-12 0 20 40 60 80 100 Donor 2 CMV Tetramer + Counts RCT-CTRL RCT-CMV RCT-CMV-4-1BBL RCT-CMV-4-1BBL-IL-12 0 1 10 4 2 10 4 3 10 4 Donor 3 CMV Tetramer + Counts RCT-CTRL RCT-CMV RCT-CMV-4-1BBL RCT-CMV-4-1BBL-IL-12 0 50 100 Donor 3 % Memory of Tetramer+ Naive/Tscm (CD45RO - CCR7 + ) Tcm (CD45RO + CCR7 + ) Tem (CD45RO + CCR7 - ) Temra (CD45RO - CCR7 - ) RCT-CTRL RCT-CMV RCT-CMV-4-1BBL RCT-CMV-4-1BBL-IL-12 0 100 200 300 400 500 Donor 1 Total IFN Spots/2×10 5 PBMC RCT-CTRL RCT-CMV RCT-CMV-4-1BBL RCT-CMV-4-1BBL-IL-12 0 100 200 300 400 Donor 2 Donor 2 IFN Spots/2×10 5 PBMCs RCT-CTRL RCT-CMV RCT-CMV-4-1BBL RCT-CMV-4-1BBL-IL-12 RCT-CTRL RCT-CMV RCT-CMV-4-1BBL RCT-CMV-4-1BBL-IL-12 0.0 5.0 10 2 1.0 10 3 1.5 10 3 Donor 3 Total IFN Spots/2×10 5 PBMC TNTC A B C D 0 4 8 12 0 2 10 2 4 10 2 1 10 4 2 10 4 3 10 4 4 10 4 Pmel-1 Number (Blood) Days After Tumor Injection Pmel-1 Numbers Pmel-1 Numbers in 50 μl Blood 1 10 9 mRBC-CTRL 1 10 9 2.5 10 8 6 10 7 ** ** Dose mRBC-gp100-4-1BBL-IL-12 mRBC-CTRL 1 10 9 2.5 10 8 6 10 7 0 1 10 5 2 10 5 3 10 5 4 10 5 Pmel-1 Number (Spleen) ** mRBC-gp100-4-1BBL-IL-12 mRBC-CTRL 1 10 9 2.5 10 8 6 10 7 0 1 10 5 2 10 5 3 10 5 4 10 5 Pmel-1 Number (Spleen) CD90.1 + CD8 + Number ** mRBC-gp100-4-1BBL-IL-12 0 20 40 60 80 100 CD8 Functionality (Lung) Effector + % of CD8 T cells IFN + % GzmB + % IFN + GzmB + % ** ** ** ** ** ** Dose aAPC T cell 1 10 9 1 10 9 2.5 10 8 6 10 7 1 10 9 + Ctrl + + + Pmel-1 endogenous E F G mRBC-CTRL mRBC-gp100-4-1BBL-IL-12 Take Down aAPC Dosing B16-F10 Pmel-1 D0 D1 D4 D8 D14 mRBC-CTRL 1 10 9 2.5 10 8 6 10 7 0 50 100 150 200 250 Lung Metastasis Count Lung Metastasis Count ** ** ** mRBC-gp100-4-1BBL-IL-12 A C D B D0 D4 D11 aAPC Dosing D7 OT1 D12 Take Down Antigen Specific T cells (Spleen) 1 10 9 mRBC-CTRL 1 10 9 3 10 8 6 10 7 0.0 5.0 10 2 1.0 10 3 1.5 10 3 2.0 10 3 2.5 10 3 3.0 10 3 Antigen Specific T cells (Blood) OVA Specific T cell Number in 30μl Blood OT1 endogenous OVA tetramer+ ** mRBC-OVA-4-1BBL-IL-12 1 10 9 mRBC-CTRL 1 10 9 3 10 8 6 10 7 0 2 10 3 4 10 3 6 10 3 8 10 3 1 10 4 OVA Specific T cell Number * * mRBC-OVA-4-1BBL-IL-12 OT1 endogenous OVA tetramer+ *: p<0.05 **: p<0.01 compared to mRBC-CTRL student t-test A C B B A D C 1x10 9 mRBC-CTRL 1x10 9 mRBC-OVA-4-1BBL-IL-12 3x10 8 mRBC-OVA-4-1BBL-IL-12 6x10 7 mRBC-OVA-4-1BBL-IL-12 1x10 9 mRBC-CTRL 1x10 9 mRBC-OVA-4-1BBL-IL-12 3x10 8 mRBC-OVA-4-1BBL-IL-12 6x10 7 mRBC-OVA-4-1BBL-IL-12 0 5 10 15 20 25 -10 -5 0 5 10 Body Weight Days Post Dose 1 Body Weight Change (%) Dose IFN Dose Liver Enzymes (ALT) 1x10 9 mRBC-CTRL 1x10 9 3x10 8 6x10 7 0 100 200 300 400 ALT (U/L) * Day 12 Day 25 mRBC-OVA-4-1BBL-IL-12 0 2 4 6 8 10 12 14 16 18 20 22 24 26 0 500 1000 1500 2000 2500 3000 3500 Days Post Dose 1 IFN (pg/ml) Take Down aAPC Dosing D0 D4 D7 D11 D12 D25 mRBC-CTRL 0/8 regressions mRBC-OVA-4-1BBL 2/8 regressions mRBC-OVA-4-1BBL-IL-12 5/8 regressions; 16-fold lower dose mRBC-OVA-4-1BBL-IL-12 7/8 regressions; 4-fold lower dose PBS mRBC-OVA-4-1BBL (2.5 10 8 ) mRBC-OVA-4-1BBL-IL-12 (2.5 10 8 ) mRBC-OVA-4-1BBL-IL-7 (2.5 10 8 ) mRBC-OVA-4-1BBL-IL-15 (2.5 10 8 ) 0 5 10 15 20 25 0 1000 2000 3000 4000 5000 Days After Tumor Randomization Tumor Volume (mm 3 ) Tumor Volume Dose PBS mRBC-CTRL mRBC-OVA-4-1BBL-IL-12 (4-fold lower dose) mRBC-OVA-4-1BBL-IL-12 (16-fold lower dose) mRBC-OVA-4-1BBL 0 5 10 15 20 25 30 35 40 45 0 50 100 Survival Days After Tumor Randomization Percent Survival p<0.0001 p<0.0001 Log rank test compared to mRBC-CTRL Dose D1 D4 D7 OT1 aAPC dosing D-7 EG7.OVA 0 5 10 15 20 25 0 1000 2000 3000 Days After Tumor Randomization Tumor Volume (mm 3 ) Efficacy Potency Dose C D B A mRBC-CTRL mRBC-OVA-4-1BBL-IL-12 0.0 0.2 0.4 0.6 0.8 1.0 OT1 Clone Frequency (Tumor) CASSRANYEQYF Clone % ** PBS mRBC-CTRL mRBC-OVA-4-1BBL IL-15 IL-12 IL-7 0 2 10 5 4 10 5 6 10 5 8 10 5 OT1 Phenotype (Spleen) OT1 Number Tscm Tcm Tem mRBC-OVA-4-1BBL PBS mRBC-CTRL mRBC-OVA-4-1BBL IL-15 IL-12 IL-7 0.0 5.0 10 5 1.0 10 6 1.5 10 6 OT1 Functionality (Spleen) OT1 Number IFN mRBC-OVA-41BBL GzmB IFN +GzmB+ PBS mRBC-CTRL mRBC-OVA-4-1BBL IL-15 IL-12 IL-7 0 4 10 2 1 10 4 2 10 4 5.0 10 4 1.0 10 5 1.5 10 5 OT1 Phenotype (Blood) mRBC-OVA-4-1BBL Tscm Tcm Tem PBS mRBC-CTRL mRBC-OVA-4-1BBL IL-15 IL-12 IL-7 0 1 10 5 2 10 5 3 10 5 5.0 10 5 1.0 10 6 1.5 10 6 OT1 Number OT1 Number mRBC-OVA-4-1BBL Spleen dLN Tumor D0 D3 D6 OT1 Take down aAPC dosing D-8 EG7.OVA PBS mRBC-CTRL mRBC-OVA-4-1BBL IL-15 IL-12 IL-7 0.0 1.5 10 3 3.0 10 3 5.0 10 4 1.0 10 5 1.5 10 5 OT1 Expansion (Blood) OT1 Number in 50μl Blood OT1 Number in 50μl Blood Day 0 Day 3 Day 6 mRBC-OVA-4-1BBL C B A D E F G B 0 1 10 4 2 10 4 3 10 4 4 10 4 OT1 Number Total OT1 Number C 0.0 5.0 10 2 1.0 10 3 1.5 10 3 CD122+CD44-CD62L+ # Tscm OT1 Number E 0.0 5.0 10 3 1.0 10 4 1.5 10 4 CD122+CD44+CD62L+ # Tcm OT1 Number F 0.0 5.0 10 2 1.0 10 3 1.5 10 3 2.0 10 3 CD122+CD44+CD62L- # Tem OT1 Number mRBC-CTRL mRBC-OVA-4-1BBL mRBC-OVA-4-1BBL-IL-7 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-15 D EL4 5:1 2:1 1:1 0.5:1 0 20 40 60 80 100 OT1:Target % Killing G EG7.OVA 5:1 2:1 1:1 0.5:1 0 20 40 60 80 100 OT1:Target % Killing RCT-OVA-4-1BBL RCT-OVA-4-1BBL-IL-7 RCT-OVA-4-1BBL-IL-12 RCT-OVA-4-1BBL-IL-15 A 4-1BB-L OVA specific TCR MHC I OVA peptide Signal 3 cytokine Tumor Cell Killing Activation/Expansion OT1 T cell Tumor cell EG7.OVA EL4 (parental) mRBC-aAPC (OVA) OT1 T cell