Poster #LB-082 In Vivo Efficacy and Pharmacodynamic Analysis of RTX-321, an Engineered Allogeneic Artificial Antigen Presenting Red Cell Therapeutic

Mellissa J. Nixon*, Xuqing Zhang*, Shamael Dastagir, Albert Lee, Mengyao Luo, Annie Khamhoung, Andrea Schmidt, Douglas C. McLaughlin, Viral Amin, Chris Moore, Jennifer Mellen, Laurence Turka, Thomas J. Wickham, and Tiffany F. Chen

The American Association for Cancer Research Annual Meeting/June 22-24, 2020

INTRODUCTION

The risk of cancer is strongly associated with human papillomavirus (HPV) infection, with the high-risk strain, HPV 16, accounting for approximately 70% of all cervical cancers and 80% of head and neck cancers associated with HPV infection.1,2 HPV 16-positive (HPV 16+) malignancies remain a critical area of unmet need for new and innovative treatment options.

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.

OBJECTIVES

• To demonstrate the direct interactions of a Rubius aAPC and antigen-specific cells in vitro

• To demonstrate that this interaction occurs in vivo and induces long-lasting anti-tumor immunological memory

• To demonstrate that aAPCs can promote anti-tumor immune responses and correlate pharmacodynamic changes with anti-tumor response

• To demonstrate RTX-321 expansion and activation of antigen-specific human T cells in vitro

Figure 1: The RED PLATFORM® Is Designed to Generate Allogeneic, Off-the-Shelf Cellular Therapies, Including RTX-321

MHC = major histocompatibility complex.

• The enucleated reticulocytes 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 That Expresses Signals 1+2+3 for T Cell Activation

RTX-321 consists of CD34+ stem cell–derived, allogeneic, engineered, human-enucleated red cells expressing human

leukocyte antigen (HLA)-A*02:01 and ββ2 microglobulin with HPV 16 oncoprotein E7 peptide (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.

RUBIUS THERAPEUTICS TERMINOLOGY

• RCT = experimental construct

• RTX = Red Cell Therapeutic™ product candidate

• mRBC = mouse surrogate experimental construct

TCRSignal 1 Tumor Antigen: HPV16 E7

RTX-321 RTX-321

MHC I (HLA-A2)

T Cell

Signal 3 Cytokine:

IL-12

Signal 2 Co-StimulatoryAgonist: 4-1BBL

Figure 5: mRBC-321 Treatment Significantly Expands Antigen-Specific T cells in both the Blood and Tumor, which Correlates with Polyfunctionality within the Tumor

(A) CD45.1 Pep Boy mice were inoculated subcutaneously with 2×106 EG7.OVA cells. When the tumors reached a volume of approximately 175 mm3, the animals

were randomized and treated with 1×106 naïve OT1 cells. After, 1×109 mRBC-CTRL or a dose titration of mRBC-321 (1×109, 2.5×108) was administered on Days 0 and 3.

TCRβ immunosequencing analysis was performed to determine OT1 frequency in blood on Day 0 (pre–mRBC-321 dose), 7 (post–mRBC-321 dose) and in the tumor

on Day 7. (B) OT1 productive frequency significantly increased in the post-treatment blood and within the tumor of mRBC-321–treated mice. (C) As measured by

flow cytometry, polyfunctionality (%Granzyme B+ IFNγβ+ ) in the tumor-infiltrating OT1 cells was significantly increased in mRBC-321–treated mice. Data points

represent the mean ± standard deviation of 5 mice; one-way ANOVA compared to mRBC-CTRL within timepoints; *P<0.05, **P<0.01, ****P<0.0001.

ACT = adoptive cell transfer; CTRL = control; mRBC = murine red blood cell; mRBC-321 = mRBC-OVA-4-1BBL-IL-12; neg=negative; OVA = H-2Kb-ovalbumin peptide;

TCRβ =T-cell receptor beta chain.

• mRBC-321 treatment significantly induces OT1 expansion in the blood and tumor

• OT1 cells within the tumor are polyfunctional after mRBC-321 treatment

Figure 6: mRBC-321 Promotes Immune Memory, Epitope Spreading, and Endogenous T cell Expansion

(A) CD45.1 Pep Boy mice were inoculated subcutaneously with 2×106 EG7.OVA tumor cells. When the tumors reached a volume of approximately 231 mm3, the

animals were randomized and adoptively transferred 1×106-naïve OT1 cells on Day 1. 1×109 mRBC-CTRL or 2.5×108 mRBC-321 was administered on Days 1, 4, and 7.

Tumor volumes were monitored until tumors had regressed and were stable by Day 65. (B) To determine long-term immunological memory, mRBC-321–cured mice

were rechallenged with 2 x 106 EG7.OVA tumor cells in addition to age-matched, naïve control mice with OT1 ACT. mRBC-321 cured–mice that were protected from

EG7.OVA rechallenge were then inoculated with EL4 tumors (the OVA-negative parental cell line) in addition to age-matched, naïve control mice. (C) Antigen-specific

OT1 T cells and endogenous OVA-specific T cells (OVA tetramer+) were significantly expanded in circulation on Day 76, 10 days after EG7.OVA rechallenge. (D) TCRβ

sequencing was performed on gDNA from blood collected prior to EG7.OVA rechallenge (Day 65), after rechallenge (Day 73) and prior to EL4 challenge (Day 126)

and after challenge (Day 136). The top TCR clones expanded after EL4 challenge were tracked throughout the tumor challenges and plotted as the sum of the clone

frequencies in complete responders (mice with no measurable EL4 tumor burden), partial responders (mice with delayed tumor growth compared to naïve, and

non-responders (mice with tumor volumes no different from naïve). Data points represent individual mice (B) or mean ± standard deviation (C); RM one-way ANOVA or

paired t-test where appropriate compared to Day 64. *P<0.05.

ACT = adoptive cell transfer; CTRL = control; Day = days after initial treatment randomization; mRBC = murine red blood cell; mRBC-321 = mRBC-OVA-4-1BBL-IL-12;

ND = not determined; OVA = H-2Kb-ovalbumin peptide; TCRββ = TCRβ β-chain.

• Mice treated with mRBC-321 were cured of their EG7.OVA tumor burden

• mRBC-321–cured mice were completely protected from EG7.OVA rechallenge

• Expansion of both OT1 and endogenous OVA-specific T cells in circulation correlated with an anti-tumor response to the initial tumor, as well as protection against tumor rechallenge

• mRBC-321–cured mice were partially protected from parental OVA-negative EL4 tumor challenge, demonstrating epitope spreading

Figure 3: The Mouse Surrogate mRBC-321 Directly Interacts with Antigen-Specific T Cells to Promote Expansion In Vitro

(A) Representative frame of confocal live-cell imaging analyses of AF488-H57 Fab labeled activated OT1 T cells (green) within 10 to 30 minutes after landing onto a

layer of immobilized CellTrace™ Far Red-labeled mRBC-CTRL or mRBC-321 chemically conjugated with DL650-4-1BBL (red), (B) OT1 cells significantly and

preferentially colocalize with mRBC-321 using Mander’s colocalization coefficients across the still frames of a live-cell imaging video. (C) After 3 days of co-culture

with mRBCs, OT1 cells significantly expand in response to mRBC-321 over mRBC-CTRL. Data points represent the mean ± standard deviation of 54 to 58 frames (B)

or mean counts ± standard deviation in 6 replicates (C). ****P<0.0001.

AF488-H57 Fab = Anti-Mouse TCR (βchain) antibody (clone H57) Fab fragment conjugated to Alexa Fluor™ 488; CTFR = CellTrace™ Far Red-labeled; CTRL = control;

DL650-4-1BBL = 4-1BBL protein conjugated to DyLight™ 650; IL = interleukin; mRBC = murine red blood cell; mRBC-321 = mRBC-OVA-4-1BBL-IL-12;

OVA = H-2Kb-ovalbumin peptide.

• mRBC-321 directly interacts with OT1 cells as demonstrated by confocal live cell imaging

• This direct interaction leads to significant expansion of OT1 cells in vitro

Figure 4: mRBC-321 Interacts with and Activates OT1 T Cells in the Spleen

(A) CD45.1 Pep Boy mice were adoptively transferred with 2×106 naïve CellTrace™ Yellow-labeled OT1 cells before dosing with 1×109 CellTrace™ Far Red-labeled

mRBC-CTRL, or mRBC-321. One hour or 17 hours post-mRBC injection, mice were sacrificed and tissues were processed into OCT blocks for immunofluorescence

analyses. (B) mRBCs per tissue area were quantified for all major organs. (C) OT1 T cells significantly co-localized with mRBC-321 in the spleen and (D) were

quantified with HALO Object Colocalization FL v1.0 software module. Spleens were harvested and analyzed by flow cytometry. (E) OT1 cells preferentially formed

doublets with mRBC-321 by flow cytometry, which correlated with (F) an increased number of OT1 cells expressing CD44, a marker of T cell activation. Data are

represented as mean ± standard deviation of individual mice (n=5). One-way ANOVA with Tukey’s multiple comparisons was performed on (B) within tissues and a

t-test was performed within timepoints (D, E and F). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

ACT = adoptive cell transfer; CTFR = CellTrace™ Far Red-labeled; CTRL = control; CTY = CellTrace™ Yellow-labeled; Hr = hours after mRBC dosing; LN = lymph node;

mRBC = murine red blood cell; mRBC-321 = mRBC-OVA-4-1BBL-IL-12; OVA = H-2Kb-ovalbumin peptide.

• mRBC-321 preferentially localized to spleen shortly after dosing

• OT1 T cells significantly colocalized and formed doublets with mRBC-321 compared to mRBC-CTRL in the spleen

• mRBC-321 induces OT1 T cell activation in the spleen

Figure 7: mRBC-321 Inhibits Tumor Growth Without OT1 Transfer, which is Correlated with Increased Endogenous OVA-Specific T Cells in the Blood and Tumor

(A) C57BL/6J mice were inoculated subcutaneously with 2×106 EG7.OVA cells, and animals were randomized and treated the next day. 1×109 mRBC-CTRL or a dose titration of mRBC-321 (1×109 or 3×108) was administered on Days 1, 4, 8, and 11. (B) Tumor volumes were monitored every 2-3 days and mRBC-321, at both dose levels,

demonstrated delay in tumor growth represented as (B) average tumor volume. (C) On Day 15, there was >18-fold increase in OVA–antigen-specific T cells within the tumor, as well as >10-fold increase in infiltration of non–OVA-specific CD8 T cells within the tumor with mRBC-321 treatment. (D) mRBC-321 treatment significantly

induced markers of proliferation (Ki67), memory formation (TEM), and activation (KLRG1 and PD-1) and effector function (GranzymeB) on CD8 T cells in circulation on Day 15, (E) which correlated with increased IFNγβ secretion overtime (E). Data points represent the mean ± standard deviation of 8 mice and n=4 for mRBC-CTRL,

n=6 for 3×108 mRBC-321 and n=5 for 1×109 mRBC-321 for the tumor analysis; one-way ANOVA with a Dunnett’s multiple comparison was calculated on Day 15 (B and D) and a RM two-way ANOVA with a Dunnett’s increased comparison was used in (F) *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

CTRL = control; Day = days after treatment randomization; KLRG1 = Killer cell lectin-like receptor subfamily G member 1; mRBC = murine red blood cell; mRBC-321 = mRBC-OVA-4-1BBL-IL-12; NEG = negative; OVA = H-2Kb-ovalbumin peptide; OVA tet = H-2Kb-ovalbumin peptide tetramer; POS = positive; RM = repeated measures;

TEM = effector memory T cell.

• mRBC-321 significantly inhibited tumor growth without antigen-specific T cell adoptive transfer

• OVA-specific T cells expanded in the tumor after mRBC-321 treatment

• mRBC-321 induced non–OVA-specific CD8 T cell infiltration into tumors

• mRBC-321 increased proliferation, memory formation, activation and effector function of CD8 T cells in circulation

Figure 8: RTX-321 Induces Expansion and Activation of HPV–Antigen-Specific T Cells In Vitro

CD8 T cells from human donors were transduced with lentivirus to express HPV E7-specific TCR with ~20-25% TCR expression (E7 TCR-T). E7 TCR-T cells and untransduced CD8 T cells were incubated with RCT-CTRL, RCT-HPV, RCT-HPV-4-1BBL, RCT-CMV-4-1BBL-IL-12 (a negative control), or RTX-321. After 24 hours (A), CD69 was

significantly upregulated on RCT-HPV, RCT-HPV-4-1BBL, and RTX-321, indicating TCR engagement. (B) At 24 hours, GranzymeB was significantly induced in E7 TCR-T cells after incubation with RTX-321, as determined by flow cytometry. (C) On Day 5, fold expansion of E7 TCR-T CD8 T cells was calculated over media control by flow

cytometry and (D) IFNγβ secretion into the supernatant was determined by cytokine multiplex array. Data points represent the mean ± standard deviation of technical duplicates of E7 TCR-T cells from 3 donors; one-way ANOVA compared to RCT-CTRL ***P<0.001, ****P<0.0001.

4-1BBL = 4-1BB ligand; CD69 = Cluster of differentiation 69, CTRL = control; E7 TCR-T = primary T cells transduced with a lentivirus to express HPV E7-specific TCR with ~20-25% TCR expression; GZMB = Granzyme B; HPV = human leukocyte antigen A2-human papillomavirus E7 peptide; IL = interleukin; RCT = experimental construct; RTX-321

= RTX-HPV-4-1BBL-IL-12 product candidate; TCR = T-cell receptor.

• While signal 1 alone is sufficient for TCR engagement, all 3 signals are required for robust expansion of E7 TCR-T cells

• Control RCT-CMV-4-1BBL-IL-12 does not expand HPV E7 antigen-specific CD8 T cells, indicating the requirement of cognate signal 1

• Presentation of HPV-signal 1 on MHC I induces early activation by upregulation of CD69 specifically on HPV E7 antigen-specific cells and not untransduced CD8 T cells

• All 3 signals are required on RTX-321 to induce a robust IFNγ and GranzymeB production

CONCLUSIONS

• mRBC-321, the mouse surrogate of RTX-321, directly interacts with antigen-specific OT1 cells in vitro as well as in the spleen in vivo, leading to expansion and activation of OT1 T cells

• mRBC-321–induced OT1 expansion can be detected in both the circulation and within the tumor, which is correlated with induction of polyfunctionality within the tumors

• mRBC-321 treatment leads to EG7.OVA tumor cures and long-term memory based on protection from rechallenge with EG7.OVA tumor cells

• Protection of mice challenged with parental EL4 cells lacking OVA strongly suggests treatment with mRBC-321 promotes epitope spreading

• mRBC-321 inhibits tumor growth without OT1 adoptive transfer, which is correlated to endogenous OVA-specific T cell expansion in the tumor

• RTX-321 activates and expands HPV-antigen–specific TCR-transduced primary T cells in vitro

• Overall, mRBC-321 and RTX-321 can selectively engage and activate antigen-specific T cells, allowing for robust expansion and differentiation into effector and long-lasting anti-tumor memory cells

• Taken together, these findings support the potential of RTX-321 as an effective therapy for antigen-specific HPV 16+ cancers. Rubius plans to file an Investigational New Drug application by the end of 2020

ACKNOWLEDGEMENTS & DISCLOSURES

Asterisks next to author names denote equal contribution.

ALL AUTHORS: Employment with and equity ownership in Rubius Therapeutics.

REFERENCES

1Saraiya M, et al. US assessment of HPV types in cancers: implications for current and 9-valent HPV vaccines. J Natl Cancer Inst. 2015;107(6):djv086.2Ndiaye 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.

Alexa Fluor, CellTrace, and DyLight are trademarks of Thermo Fisher Scientific.

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