Poster #F290 An Engineered Allogeneic Artificial Antigen-Presenting Red Cell Therapeutic™, RTX-321, Promotes Antigen-Specific T Cell Expansion and Anti-Tumor Activity Xuqing Zhang, Mengyao Luo, Shamael R Dastagir, Mellissa Nixon, Annie Khamhoung, Andrea Schmidt, Billy Blanco, Viral Amin, Ryan Pepi, Timothy J Lyford, Laurence A Turka, Thomas J Wickham, and Tiffany F Chen FOCIS Virtual Annual Meeting/October 28-31, 2020 INTRODUCTION Human papillomavirus (HPV) 16 is associated with approximately 70% of cervical cancers, approximately 40% of head and neck squamous cell carcinoma (HNSCC) arising in the oropharynx, approximately 25-40% of HNSCC arising in other locations, and approximately 80%-85% of anal cancers. 1-3 Despite available therapies, a critical need remains for new treatment options for advanced HPV 16-associated cancers. Red Cell Therapeutics™ (RCTs™) are a new class of allogeneic, off-the-shelf cellular therapeutic candidates for the treatment of cancer and autoimmune diseases. RCTs are engineered to mimic human immunobiology and induce a tumor-specific immune response by expanding tumor-specific T cells against a target antigen in vivo. Rubius Therapeutics’ first artificial antigen-presenting cell (aAPC) product candidate, RTX-321, is for the treatment of HPV 16+ cancers. Figure 1: The RED PLATFORM ® is Designed to Generate Allogeneic, Off-the-Shelf Cellular Therapies MHC=major histocompatibility complex. • The enucleated red blood cells are RCTs that express hundreds of thousands of biotherapeutic proteins on the cell surface • Universal, scalable, and consistent manufacturing process Figure 2: RTX-321 is a Cellular Therapy With a Dual Mechanism RTX-321 consists of allogeneic, cultured, human-enucleated red blood cells engineered to express HPV 16 oncoprotein E7 peptide presented on human leukocyte antigen (HLA)-A*02:01 and β2 microglobulin (HLA-A2-HPV; Signal 1), 4-1BB ligand (4-1BBL; tumor necrosis factor superfamily member 9; Signal 2), and a fusion protein of interleukin-12 (IL-12; Signal 3) p40 and p35 subunits on the cell surface. MHC=major histocompatibility complex; RTX-321=RTX-HPV-4-1BBL-IL-12 product candidate; TCR=T cell receptor. OBJECTIVES • To demonstrate the anti-tumor efficacy, long-term anti-tumor memory, and epitope spreading by RTX-321 mouse surrogates • To demonstrate the dual mechanism of action of RTX-321 and its mouse surrogates: – Boosts target antigen-specific CD8+ T cell responses as an artificial antigen-presenting cell (aAPC) – Promotes target antigen-independent adaptive and innate immune responses Figure 3: Tumor Models for Efficacy Evaluation of RTX-321 Mouse Surrogates (A) mRBC-gp100-4-1BBL-IL-12 is conjugated with a gp100 peptide presented on H-2D b (murine MHC I, signal 1), murine 4-1BBL (signal 2), and murine IL-12 (signal 3) to activate gp100-specific pmel-1 transgenic CD8+ T cells. These pmel-1 T cells target gp100 expressing B16-F10 melanoma tumor cells. (B) mRBC-OVA-4-1BBL-IL-12 is conjugated with an OVA peptide presented on H-2K b (murine MHC I, signal 1), murine 4-1BBL (signal 2), and murine IL-12 (signal 3) to activate OVA-specific OT-1 transgenic CD8+ T cells. These OT-1 T cells target OVA expressing EG7.OVA lymphoma tumor cells. 4-1BBL=4-1BB ligand; gp100 peptide=glycoprotein 100 peptide KVPRNQDWL; IL=interleukin; mRBC=murine red blood cell; OVA peptide=ovalbumin peptide SIINFEKL; TCR=T cell receptor. Figure 4: mRBC-aAPCs Targeted Against a Tumor-Associated Antigen (gp100) Promote Antigen-Specific pmel-1 T Cell Expansion and Effector Function and Dramatically Reduce Lung Metastasis of B16-F10 Tumors (A) C57BL/6 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 (n=5 to 8) were then dosed with 1×10 9 mRBC-CTRL, 1×10 9 mRBC-CTRL with αPD-1, 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 mice dosed with 1×10 9 mRBC-CTRL, 1×10 9 mRBC-CTRL+αPD-1, or 1×10 9 mRBC-gp100-4-1BBL-IL-12. (C) Lung metastasis counts on Day 14. (D-G) pmel-1 cell number in 50μL blood (D) the spleen (E) the left lobe of perfused lung (F) and the effector function of lung infiltrating pmel-1 and endogenous CD8+ T cells (G). Data are depicted as mean ± s.d.; One-way ANOVA compared to mRBC-CTRL; **P< 0.01, ****P< 0.0001. 4-1BBL=4-1BB ligand; aAPC=artificial antigen-presenting cell; PD-1=anti-programmed cell death protein 1; CTRL=control; D=Day; gp100=H-2D b -gp100; IFNγ=interferon γ; IL=interleukin; mRBC=murine red blood cell. • mRBC-gp100-4-1BBL-IL-12 dramatically reduces B16-F10 lung metastasis, while check point inhibitor αPD-1 had no effects • 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 5: mRBC-OVA-4-1BBL-IL-12 Inhibits EG7.OVA Tumor Growth With and Without Adoptive T Cell Transfer of OT-1 T Cells and Significantly Increases OVA-specific T Cells in Tumors (A) CD45.1 Pep Boy mice were inoculated subcutaneously with 2×10 6 EG7.OVA cells and randomized when tumors reached ~230 mm 3 (n=8), treated with 1×10 6 naïve OT-1 cells, and dosed with 1×10 9 mRBC-CTRL or a dose titration of mRBC-OVA-4-1BBL-IL-12 (1×10 9 , 2.5×10 8 ). (B) Tumor growth curve after randomization and treatments. (C) In a separate study, CD45.1 Pep Boy mice were inoculated subcutaneously with 2×10 6 EG7.OVA cells and randomized when tumors reached ~175 mm 3 (n=5), treated with 1×10 6 naïve OT-1 cells, and dosed with 1×10 9 mRBC-CTRL or a dose titration of mRBC-OVA-4-1BBL-IL-12 (1×10 9 , 2.5×10 8 ). (D) OT-1 numbers per gram of tumor on Day 7. (E) In a separate study, C57BL/6 mice were inoculated subcutaneously with 2×10 6 EG7.OVA cells. Subsequently, 1×10 9 mRBC-CTRL, or a dose titration of mRBC-OVA-4-1BBL-IL-12 (1×10 9 , 3×10 8 ) was administered (n=8) on Days 1, 4, 8, and 11. (F) Tumor growth curves after randomization and treatments. (G) Representative flow plots (1×10 9 dose groups) and quantification of tumor-infiltrating OVA tetramer+ CD8+ T cells and OVA tetramer- CD8+ T cells on Day 15. Data are depicted as mean ± s.d.; One-way ANOVA compared to mRBC-CTRL, **P< 0.01, ***P< 0.001, ****P< 0.0001. 4-1BBL=4-1BB ligand; aAPC=artificial antigen-presenting cell; CTRL=control; D=Day; IL=interleukin; mRBC=murine red blood cell; OVA=H-2K b -OVA. • mRBC-OVA-4-1BBL-IL-12 demonstrated tumor growth inhibition in an EG7.OVA tumor model with and without OT-1 adoptive cell transfer • mRBC-OVA-4-1BBL-IL-12 anti-tumor effects correlated with increased OVA-specific T cells and other CD8+ T cells in the tumors Signal 1 Tumor Antigen: HPV 16 E7 RTX-321 MHC I (HLA-A2) Antigen-Specific T Cell T Cell NK Cell Signal 3 Cytokine: IL-12 Signal 2 Co-Stimulatory Agonist: 4-1BBL Figure 6: mRBC-OVA-4-1BBL-IL-12 Promotes Adaptive and Innate Immune Responses in the Tumors of EG7.OVA Bearing Mice With OT-1 Adoptive Transfer (A) CD45.1 Pep Boy mice were inoculated subcutaneously with 2×10 6 EG7.OVA cells. When the tumors reached a volume of ~175 mm 3 , the animals were randomized (n=5) and treated with 1×10 6 naïve OT-1 cells. After, 1×10 9 mRBC-CTRL or a dose titration of mRBC-OVA-4-1BBL- IL-12 (1×10 9 , 2.5×10 8 ) was administered on Days 0 and 3. Mice were sacrificed on Day 7. (B) Polyfunctionality (granzymeB+IFNγ+ %) in the tumor-infiltration OT-1 cells on Day 7. (C) Ki67, TNFα, and IL-2 per cell expression in tumor infiltrating endogenous CD8+ T cells; (D) Treg% and IFNγ+Ki67+% in tumor infiltrating CD4+ T cells; (E) Ki67, and granzymeB per cell expression in tumor infiltration NK cells; and (G) M1 macrophage % in tumor infiltrating leukocytes. Data are depicted as mean ± s.d; One-way ANOVA compared to mRBC-CTRL; *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001. 4-1BBL=4-1BB ligand; aAPC=artificial antigen-presenting cell; CTRL=control; D=Day; IFN γ=interferon γ; IL=interleukin; mRBC=murine red blood cell; M1 macrophage=M1-type classically-activated pro-inflammatory macrophages; MFI=mean fluorescence intensity; NK=natural killer cells; OVA=H-2K b -OVA; Th1=T helper 1 CD4+ T cells; TNFα=tumor necrosis factor; Treg=regulatory T cells. • mRBC-OVA-4-1BBL-IL-12 increased polyfunctional OVA antigen-specific T cells in the tumors • mRBC-OVA-4-1BBL-IL-12 promoted adaptive immune responses in the tumor as demonstrated by increased proliferation and effector cytokine expression in CD8+ T cells as well as decreased Treg and increased proliferating Th1 CD4+ T cells • mRBC-OVA-4-1BBL-IL-12 promoted the innate immune responses in the tumors as demonstrated by increased proliferation and effector molecule expression in NK cells and increased M1 macrophages Figure 7: mRBC-OVA-4-1BBL-IL-12 Boosts Broad Adaptive and Innate Immune Responses in Circulation Without Adoptive Transfer in EG7.OVA Tumor Models (A) C57BL/6 mice were inoculated subcutaneously with 2×10 6 EG7.OVA cells. Subsequently, 1×10 9 mRBC-CTRL, or a dose titration of mRBC-OVA-4-1BBL-IL-12 (1×10 9 , 3×10 8 ) was administered (n=8) on Days 1, 4, 8, and 11 (noted with arrows in B-E). (B) Ki67+%, T EM %, and granzymeB+% in CD8+ T cells; (C) Ki67+% in NK cells; (D) MHC-II+% of monocytes in the blood over time. (E) Plasma IFNγ and soluble 4-1BB concentrations over time. Data are depicted as mean ± s.d; Two-way ANOVA repeated measures with Dunnett’s multiple comparison at each time point compared to mRBC-CTRL; *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001. 4-1BBL=4-1BB ligand; aAPC=artificial antigen-presenting cell; CTRL=control; D=Day; IFNγ=interferon γ; IL=interleukin; MHC-II=major histocompatibility complex class II; mRBC=murine red blood cell; NK=natural killer cells; OVA=H-2K b -OVA; T EM =effector memory T cells. • Without adoptive transfer, mRBC-OVA-4-1BBL-IL-12 increased proliferation, effector molecule expression and effector memory formation of CD8+ T cells in circulation • mRBC-OVA-4-1BBL-IL-12 increased NK cell proliferation and MHC-II+ monocytes in circulation • mRBC-OVA-4-1BBL-IL-12 significantly increased plasma IFNγ levels • mRBC-OVA-4-1BBL-IL-12 increased soluble 4-1BB shedding, indicating 4-1BB receptor engagement Figure 8: Rechallenge of Cured Mice with EG7.OVA or Parental EL4 Tumors Demonstrates Maintenance of Memory and Epitope Spreading (A) CD45.1 Pep Boy mice were randomized when EG7.OVA tumors reached ~230 mm 3 (n=8), treated with 1×10 6 naïve OT-1 cells, and dosed with 2.5×10 8 mRBC-OVA-4-1BBL-IL-12. Seven out of 8 mice were cured of original EG7.OVA tumors and were rechallenged on Day 66 with EG7.OVA. Age-matched naïve CD45.1 Pep Boy mice (n=5) were treated on Day 65 with 5×10 5 naïve OT-1 cells one day before challenge with EG7.OVA cells, as controls. (B) All previously cured mice rejected EG7.OVA rechallenge. (C) Representative flow cytometry plots showing OT-1 and endogenous OVA-specific T cells in 50μL of peripheral blood 10 days after EG7.OVA rechallenge (Day 76). (D) OT-1 and (E) endog- enous OVA-specific T cell numbers in 50μL peripheral blood 2 days before rechallenge (Day 64), 4 days post rechallenge (Day 70), and 10 days post rechallenge (Day 76). (F) At 61 days post-second EG7.OVA challenge (Day 127), cured mice (n=7) along with age-matched naïve control mice (n=5) were challenged with EL4. Three out of 7 cured mice had delayed EL4 growth and 3 out of 7 rejected EL4. (G) TCR sequencing analyses of OT-1 frequency on Days 65, 73, 126, and 136 in the blood. (H) The significantly expanded TCR clones after EL4 challenge were tracked by TCR sequencing throughout the tumor challenges. The sum of these clone frequencies in individual mice is shown. Data are depicted as mean ± s.d.; One-way ANOVA compared to Day 64; Student’s t-test compared to naïve; *P< 0.05, **P< 0.01, ***P< 0.001. 4-1BBL=4-1BB ligand; aAPC=artificial antigen-presenting cell; IL=interleukin; mRBC=murine red blood cell; OVA=H-2K b -OVA; TCRseq=TCR sequencing. • mRBC-OVA-4-1BBL-IL-12-dosed mice that cured EG7.OVA tumors all rejected EG7.OVA rechallenge • OT-1 cells, in addition to endogenous OVA-specific T cells, expanded after the EG7.OVA rechallenge • Challenge with the parental EL4 cell line demonstrated 3/7 delays in tumor growth and 3/7 cures, suggesting epitope spreading • OT-1 clones increased in frequency after EG7.OVA rechallenge in the blood of mRBC-OVA-4-1BBL-IL-12 cured mice • Increased frequency of EL4-responsive TCRs upon each tumor challenge (EG7.OVA and EL4) was associated with complete responders (mice that rejected EL4 challenge), suggesting that the ability to control EL4 tumors correlated with the expansion of EL4-responsive TCR clones Figure 9: mRBC-OVA-4-1BBL-IL-12 is Well Tolerated and Has a Preferential Biodistribution to the Spleen (A) CD45.1 Pep Boy mice (n=4 to 10) with or without 1×10 6 naïve OT-1 transfer were dosed with 1×10 9 mRBC-CTRL or a dose titration of mRBC-OVA- 4-1BBL-IL-12 (1×10 9 , 3×10 8 ) on Days 0, 4, 7 and 11 (noted with arrows in B and C). (B) Body weight changes compared to Day 0. (C) Plasma IFNγ levels over time. (D) Serum ALT levels on Days 12 and 25. (E) C57BL/6 mice (n=5) were dosed with 1×10 9 CellTrace TM Far Red dye-labeled mRBC-CTRL or mRBC-OVA-4-1BBL-IL-12 on Days 1 and 4. (F) Fluorescently labeled mRBCs per tissue area by immunofluorescent analyses on Day 1 (1 hour post dose 1) or on Day 8 (96 hours post dose 2). (G) mRBC-OVA-4-1BBL-IL-12 density in the liver, bone marrow and heart compared to the spleen. Data are depicted as mean ± s.d.; Two-way ANOVA repeated measures with Dunnett’s multiple comparison at each time point compared to mRBC-CTRL (B, C) or one-way ANOVA compared to mRBC-CTRL (F); *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001. Additional groups were evaluated but not reported from these studies. 4-1BBL=4-1BB ligand; aAPC=artificial antigen-presenting cell; ALT=alanine transaminase; CTRL=control; D=day; IFNγ=interferon γ; IL=interleukin; LN=lymph node; mRBC=murine red blood cell; OVA=H-2K b -OVA. • mRBC-OVA-4-1BBL-IL-12 induced minimal reversible effects in vivo, including body weight changes, plasma IFNγ levels, and serum ALT levels • mRBC-OVA-4-1BBL-IL-12 density was the highest in the spleen. mRBC-OVA-4-1BBL-IL-12 density in the liver and bone marrow was also higher than in other tissues (much lower than in the spleen). The increase in density in the heart was possibly due to assay variation and not biologically relevant • On Day 8 (96 hours after dose 2), the densities of mRBC-OVA-4-1BBL-IL-12 had declined substantially to minimal levels RESULTS C D E F G 0 50 100 150 200 250 Lung Metastasis Count Lung Metastasis Count **** **** **** mRBC-gp100-4-1BBL-IL-12 mRBC-CTRL mRBC-CTRL+ PD-1 6 10 7 2.5 10 8 1 10 9 mRBC-CTRL mRBC-CTRL+ PD-1 6 10 7 2.5 10 8 1 10 9 2 10 2 4 10 2 6 10 2 1 10 4 2 10 4 3 10 4 4 10 4 5 10 4 CD90.1 + CD8 + Number in 50μl Blood Day 4 Pmel-1 Number (Blood) Day 7 Day 11 mRBC-gp100-4-BBL-IL-12 **** **** ** Pmel-1 Number (Spleen) 0 1 10 5 2 10 5 3 10 5 4 10 5 CD90.1 + CD8 + Number **** mRBC-gp100-4-1BBL-IL-12 mRBC-gp100-4-1BBL-IL-12 0.0 5.0 10 4 1.0 10 5 1.5 10 5 Pmel-1 Number (Lung) CD90.1 + CD8 + Number **** 0 20 40 60 80 100 CD8 Functionality (Lung) Effector+% of CD8 T Cells IFN + % granzymeB + % IFN + granzymeB + % **** **** **** **** **** **** Dose aAPC T cell 1 10 9 1 10 9 2.5 10 8 6 10 7 1 10 9 + Ctrl + + + pmel-1 Endogenous 1 10 9 Ctrl αPD-1 - - - - - + mRBC-CTRL mRBC-CTRL+ PD-1 6 10 7 2.5 10 8 1 10 9 mRBC-CTRL mRBC-CTRL+ PD-1 6 10 7 2.5 10 8 1 10 9 B mRBC-CTRL mRBC-CTRL and αPD-1 A D0 D1 D4 D8 D14 B16-F10 1x10 5 Analyses 2x10 6 pmel-1 aAPC or αPD-1 Dosing mRBC-gp100-4-1BBL-IL-12 OT-1 T Cell EG7.OVA Tumor Cell IL-12 mRBC-OVA-4-1BBL-IL-12 4-1BBL OVA peptide TCR H-2K b pmel-1 T Cell B16-F10 Tumor Cell IL-12 mRBC-gp100-4-1BBL-IL-12 4-1BBL gp100 peptide TCR H-2D b RED PLATFORM ® ONE HEALTHY O- DONOR EXPANSION & DIFFERENTIATION PROGENITOR CELL COLLECTION LENTIVIRAL VECTOR ENCODING OF MHC I (HPV PEPTIDE), CO-STIMULATORY MOLECULE & CYTOKINE ENUCLEATION & MATURATION 100-1000’s OF DOSES RED CELL THERAPEUTIC Figure 10: RTX-321 Engages Primary Human HPV-Specific T Cells and Boosts Other General Adaptive and Innate Immune Responses CD8+ T cells from heathy donor PBMCs were transduced to express HPV 16 E7-specific TCR (E7 TCR-T cells). RTX-321 or RCT-CTRL (8×10 5 , 2×10 5 , 5×10 4 and/or 1.25×10 4 ) were incubated with 2×10 5 PMBCs alone or 2×10 5 PBMC+ 4×10 3 E7 TCR-T cells. On Day 5, (A) the number of HPV 16 antigen-specific CD8+ T cells, (B), HLA-DR+%, (C) granzyme B+%, (D) Tbet+% in CD8+ T cells were determined to evaluate CD8+ T cell responses. On Day 8 or 9, (E) NK cell number, (F) DNAM1+%, (G) granzymeB+% in NK cells were determined to evaluate NK cell response. (H) IFNγ concentrations in supernatant samples 9 days after RCT-CTRL or RTX-321 treatments. Data are depicted as mean ± s.d. of duplicate wells from 3 or 4 donors. Two-way ANOVA compared to RCT-CTRL; *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001. CTRL=control; DNAM1=DNAX accessory molecule; E7-TCR-T=primary CD8+ T cells transduced to express HPV 16 E7 specific TCR; HLA-DR=human leukocyte antigen DR; HPV=human papillomavirus; IFNγ=interferon γ; NK=natural killer cells; PBMCs=peripheral blood mononuclear cells; RCT=red cell therapeutics; RTX-321=RCT-HPV-4-1BBL-IL-12; Tbet=T-box expressed in T cells; TCR=T cell receptor. RTX-321 has a dual mechanism of action: • RTX-321 functions as an aAPC to boost HPV 16 antigen-specific T cells in vitro – RTX-321 selectively expands antigen-specific CD8+ T cells – RTX-321 induces activation (HLA-DR) and effector phenotype/function (Tbet and granzymeB) in CD8+ T cells in the presence of HPV 16 antigen-specific T cells – RTX-321 further increases the secretion of IFNγ in the presence of HPV 16 antigen-specific CD8+ T cells compared to PBMCs alone • RTX-321 promotes HPV 16-independent adaptive and innate immune responses in vitro – HLA-DR upregulation is observed in non-HPV 16 specific CD8+ T cells – RTX-321 expands NK cells and increases activation (DNAM1) and effector upregulation (granzymeB) in NK cells – RTX-321 increases the secretion of IFNγ in PBMCs alone CONCLUSIONS • RTX-321 and its mouse surrogates demonstrate a dual mechanism of action (1) function as an aAPC to boost antigen-specific CD8+ T cell responses, and (2) promote other target-antigen-independent stimulation of both innate and adaptive immune responses • Mouse surrogates of RTX-321 promote tumor control, memory formation and epitope spreading in tumor models in vivo • Treatment with the RTX-321 mouse surrogate results in minimal, reversible effects in vivo (body weight change, IFNγ and ALT levels). This is likely due to the biodistribution to the vasculature and spleen • Taken together, these findings support the potential of RTX-321 as an effective therapy for the treatment of HPV 16+ cancers. Rubius Therapeutics plans to file an Investigational New Drug (IND) application by the end of 2020 ACKNOWLEDGEMENTS & DISCLOSURES ALL AUTHORS: Employment with and equity ownership in Rubius Therapeutics. REFERENCES 1 Chunqing L et al. Human papillomavirus types from infection to cancer in the anus, according to sex and HIV status: a systematic review and meta-analysis. Lancet Infect Dis. 2018;18(2):198-206. 2 Saraiya M et al. US Assessment of HPV types in cancers: implications for current and 9-valent HPV vaccines. J Natl Cancer Inst. 2015 Jun; 107(6):djv086. 3 Ndiaye C et al. HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis. Lancet Oncol 2014; 15: 1319–1331. CellTrace™ is a trademark of Thermo Fisher Scientific. D OT-1 in Tumor aAPC dosing D-8 D0 D3 Analyses D7 2x10 6 EG7.OVA 1x10 6 OT-1 C B A 10 2 10 3 10 4 10 5 10 6 CD8+ T Cells in Tumor Counts/Gram Tumor **** ** **** 39-fold 11-fold Tetramer + CD8 Tetramer - CD8 ** Tumor Volume 1 10 9 mRBC-CTRL 2.5 10 8 1 10 9 CD45.2+tet+CD8+ Number/Gram Tumor mRBC-OVA-4-1BBL-IL-12 E F G 1x10 9 mRBC-CTRL 1x10 9 mRBC-OVA-4-1BBL-IL-12 2.5x10 8 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 0 5 10 15 20 0 500 1000 1500 2000 Tumor Volume mRBC-CTRL mRBC-OVA-4-1BBL-IL-12 Days After Tumor Randomization Tumor Volume (mm 3 ) OVA Tetramer ** *** D0 D1 D4 D8 D15 aAPC dosing Analyses D11 D-6 D1 D4 EG7.OVA aAPC dosing D7 2x10 6 EG7.OVA 2x10 6 1x10 6 OT-1 0 2 10 4 4 10 4 6 10 4 8 10 4 1 10 5 ** 0 5 10 15 20 25 0 500 1000 1500 2000 Days After Randomization Tumor Volume (mm 3 ) CD8 D C B A Proliferating Th1% in Tumor aAPC dosing D-8 D0 D3 Analyses D7 2x10 6 EG7.OVA 1x10 6 OT-1 GranzymeB+IFNγ+% 0 2000 4000 6000 8000 Ki67 CD8 in Tumor *** TNFα CD8 in Tumor 0 5000 10000 15000 * IL-2 CD8 in Tumor 0 200 400 600 *** 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 0 10 20 30 40 50 Treg% CD4 in Tumor ** 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 Polyfunctional OT-1% in Tumor 0 20 40 60 80 100 ** mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 1x10 9 mRBC-CTRL 1x10 9 2.5x10 8 0 20 40 60 80 **** ** 0 500 1000 1500 Ki67 NK in Tumor Ki67 MFI of NK1.1+CD3- * GranzymeB NK in Tumor GranzymeB MFI of NK1.1+CD3- 0 500 1000 1500 2000 2500 ** 0 10 20 30 40 M1 Macrophage % in Tumor MHCII+F4/80+CD11b+Ly6C-/low Ly6G-CD4-CD8-% of CD45.1+ * ** mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 Foxp3+CD25+% of CD4+CD45.1+ IFN +Ki67+% of CD4+CD45.1+ Ki67 MFI of CD8+CD45.1+CD45.2- TNFα MFI of CD8+CD45.1+CD45.2- IL-2 MFI of CD8+CD45.1+CD45.2- E F G 1x10 9 mRBC-CTRL 1x10 9 mRBC-OVA-4-1BBL-IL-12 2.5x10 8 mRBC-OVA-4-1BBL-IL-12 CD8 T Cell Response Antigen-Specific Response Adaptive Immune Response Innate Immune Response Adaptive Immune Response CD4 T Cell Response NK Cell Response Macrophage Response B A 0 5 10 15 0 20 40 60 80 100 % Ki67 CD8+ T Cells Days % Ki67+/CD8+ **** **** ** *** *** **** * 0 5 10 15 0 20 40 60 80 % T EM CD8+ T Cells Days % CD44+/CD62L neg /CD8+ **** **** * **** ** 0 5 10 15 0 20 40 60 80 % GranzymeB CD8+ T Cells Days % granzymeB+/CD8+ **** **** ** ** *** D %MHC-II+ of Monocyte 0 5 10 15 0 10 20 30 40 Days %IA-IE+/CD11b+ **** *** ** *** **** * ** *** 0 5 10 15 0 5000 10000 15000 Soluble 4-1BB Days pg/mL ** **** * ** * C 0 5 10 15 0 50 100 % Ki67 of NK Cells Days % Ki67+/NK1.1+/CD3 neg **** **** ** **** **** **** **** **** 0 5 10 15 0 500 1000 1500 2000 IFN Days pg/mL *** ** * ** ** ** ** E D0 D1 D4 D8 D15 EG7.OVA aAPC dosing Analyses D11 2x10 6 Innate Immune Response Soluble Factors CD8 T Cell Response 1x10 9 mRBC-CTRL 1x10 9 mRBC-OVA-4-1BBL-IL-12 2.5x10 8 mRBC-OVA-4-1BBL-IL-12 A 2x10 6 EG7.OVA 2x10 6 EG7.OVA Days 0 1 4 7 64 65 66 70 73 76 126 127 136 1x10 5 EL4 OT-1 aAPC TCRseq Tetramer TCRseq Randomization G F OT-1 Frequency in Blood Day 65 Day 73 Day 126 Day 136 0.0 0.1 0.2 0.3 0.4 0.5 CASSRANYEQYF Frequency Treatment Naïve (No EL4 Inoculation) mRBC-OVA-4-1BBL-IL-12 Cured EG7.OVA Rechallenge EL4 Challenge EL4 Challege Tumor Volume (mm 3 ) 0 10 20 30 0 2000 4000 6000 Days Post EL4 Challenge 1x10 5 EL4 3/7 delayed 3/7 cures Treatment Naïve (n=5) mRBC-OVA-4-1BBL-IL-12 Previously Cured (n=7) C OVA-Specific T Cell Expansion Day 10 Post Rechallenge (Blood) OT-1 Endogenous OVA Specific T Cells OVA Tetramer CD45.2=OT-1 B EG7.OVA Rechallenge OT-1 (5x10 5 ) Naive Group 0 10 20 30 40 0 2000 4000 6000 Days Post EG7.OVA Rechallenge Tumor Volume (mm 3 ) 2x10 6 EG7.OVA Rechallenge 7/7 cures Treatment Naïve (n=5) mRBC-OVA-4-1BBL-IL-12 Previously Cured (n=7) D OT-1 in Blood CD45.2+tetramer+number Day 64 Day 70 Day 76 0 1000 2000 3000 Treatment Naïve mRBC-OVA-4-1BBL-IL-12 Cured ND * *** E Endogenous OVA-Specific in Blood Day 64 Day 70 Day 76 0 500 1000 1500 2000 CD45.2-tetramer+ number * ** ND H Sum Frequency of EL4 Expanded Clones Day 65 Day 73 Day 126 Day 136 10 -5 10 -4 10 -3 10 -2 10 -1 Log (Clone Frequency) Non-Responder (1/7) Partial Responder (3/7) EG7.OVA Rechallenge EL4 Challenge Complete Responder (3/7) B A Tissue mRBC-OVA-4-1BBL-IL-12 Spleen 8813.1 100% Liver 228.2 2.6% Bone Marrow 350.7 4.0% Heart 27.9 0.3% Average Density (cells/mm 2 ) Density as a % of Spleen D C E F G D1 D4 1h D8 aAPC Dosing Analyses D0 D4 D11 D7 D12 D25 OT-1 aAPC Dosing Analyses 1x10 6 1x10 9 mRBC-CTRL+OT-1 1x10 9 mRBC-OVA-4-1BBL-IL-12+OT-1 3x10 8 mRBC-OVA-4-1BBL-IL-12+OT-1 1x10 9 mRBC-CTRL 1x10 9 mRBC-OVA-4-1BBL-IL-12 3x10 8 mRBC-OVA-4-1BBL-IL-12 Body Weight 0 5 10 15 20 25 -10 -5 0 5 10 Days Post Dose 1 Body Weight Change (%) * * 1x10 9 mRBC-CTRL 3x10 8 1x10 9 0 100 200 300 400 Liver Enzymes (ALT) ALT (U/L) Day 12 + OT-1 Day 25 + OT-1 mRBC-OVA-4-1BBL-IL-12 Day 12 Day 25 0 2 4 6 8 10 12 14 16 18 20 22 24 26 0 1 10 3 2 10 3 3 10 3 4 10 3 5 10 3 Days Post Dose 1 pg/ml IFN **** **** *** **** * **** * Spleen Liver Lung Bone marrow Mesenteric LN Mandibular LN Thymus Adrenal gland Heart Kidney Brain Stomach Intestine Muscle Testis 0 3000 6000 9000 12000 Biodistribution 7.9 fold **** mRBC-CTRL 1 Hour mRBC-OVA-4-1BBL-IL-12 1h mRBC-CTRL Day 8 mRBC-OVA-4-1BBL-IL-12 Day 8 *** **** ** 1 Hour Day 8 0 100 200 300 400 500 Bone Marrow **** 1 Hour Day 8 0 20 40 60 Heart mRBC-CTRL mRBC-OVA-4-1BBL-IL-12 ** 1 Hour Day 8 0 100 200 300 400 Liver mRBC/mm 2 mRBC/mm 2 mRBC/mm 2 mRBC/mm 2 *** B A D C HLA-DR+% of Live Total CD8 HLA-DR+% of live total CD8 0 20 40 60 80 * ** **** **** * ** F E G H NK Cell Response Cytokine CD8+ T Cell Response 0 1000 2000 3000 4000 5000 * *** *** 0 5000 10000 15000 ** **** *** 0 20 40 60 80 100 *** **** **** *** **** **** 40 60 80 100 **** *** *** *** ** ** 0 50000 100000 150000 200000 250000 **** ** * HPV 16 Antigen-Specific CD8+ T Cell Number CD8+ Tetramer+ Number NK Number CD3-CD56+ Number DNAM1+% in NK Cells DNAM1+% of CD56+ NK GranzymeB+% in NK Cells IFNγ Secretion GranzymeB+% of CD56+ NK Cells IFNγ pg/ml 0 10 20 30 40 ** **** **** Tbet+% of Live Total CD8 GranzymeB+% in CD8+ T Cells Tbet+% in CD8+ Cells 0 20 40 60 80 100 * ** *** GranzymeB+% of Live Total CD8 PBMCs alone, RCT-CTRL treated PBMCs alone, RTX-321 treated PBMC + E7 TCR-T cells, RTX-CTRL treated PBMC + E7 TCR-T cells, RTX-321 treated