Poster #321 An Engineered, Allogeneic, Artificial Antigen-Presenting Red Cell Therapeutic, RTX-321, for HPV 16+Associated Cancers Promotes Antigen-Specific T Cell Activation & Expansion

Shamael R. Dastagir, Xuqing Zhang, Albert Lee, Timothy J. Lyford, Jennifer M. Mellen, Andrea Schmidt, Mary Gribble, Chris Moore, Thomas J. Wickham, and Tiffany F. Chen

The American Society of Gene & Cell Therapy/May 12-15, 2020

INTRODUCTION

Red Cell Therapeutics™ (RCTs) are a new class of allogeneic, off-the-shelf cellular therapeutic candidates for the treatment of cancer and autoimmune diseases.

For the treatment of cancer, 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 potential treatment of HPV 16-positive cancers (HPV 16+ cancers).

OBJECTIVES

• To generate an allogeneic, artificial antigen-presenting cell with the RED PLATFORM®, RTX-321, and promote robust HPV-specific T-cell mediated responses

• To demonstrate that RTX-321 induces activation of HPV antigen-specific primary CD8+ T cells

• To demonstrate that RTX-321 selectively expands HPV antigen-specific primary CD8+ T cells

• To demonstrate that RTX-321 induces memory formation and effector molecule expression of HPV antigen-specific primary CD8+ T cells

RUBIUS TERMINOLOGYRCT = experimental construct; RTX = Red Cell Therapeutic product candidate.

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

• 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 human papillomavirus (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.

Antigen-Specific 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: RTX-321 Induces Activation Markers 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 were incubated with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12, RCT-4-1BBL-IL-12, RCT-HPV-4-1BBL, RCT-CMV-4-1BBL-IL-12,

or RTX-321. On Day 5, (A) 4-1BB+ percent (B) CD25+ percent and (C) PD-1+ percent of CD8 T cells were determined by flow cytometry to evaluate

T cell activation. Data points represent the mean of technical duplicates of E7 TCR-T cells from 3 donors; error bars represent standard

deviation; One-way ANOVA compared to RCT-CTRL, *P< 0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

4-1BB = tumor necrosis factor superfamily member 9; 4-1BBL = 4-1BB ligand; CD25 = cluster of differentiation 25; CTRL = control; HLAA2 = human

leukocyte antigen A2; HPV = HLA-A2-human papillomavirus E7 peptide; IL = interleukin; PD-1 = programmed cell death protein 1; RCT= Red Cell

Therapeutic experimental construct; RTX-321 = RTX-HPV-4-1BBL-IL-12 product candidate; TCR = T cell receptor.

• 4-1BB upregulation is driven by signal 1 on RCT-HPV, RCT-HPV-4-1BBL, and RTX-321

• CD25 upregulation is driven by all 3 signals presented on RTX-321

• PD-1 upregulation is driven by signal 1 but enhanced with all 3 signals presented on RTX-321

Figure 6: RTX-321 Expands HPV-Specific T Cells

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-4-1BBL, RCT-IL-12, RCT-4-1BBL-IL-12,

RCT-HPV-4-1BBL, RCT-CMV-4-1BBL-IL-12, or RTX-321. E7 TCR+ expression of (A) RCT-CTRL and (B) RTX-321 treated E7 TCR-T cells was

determined by HPV E7-specific tetramer staining on Day 5. Fold expansion of (C) E7 TCR-T and (D) untransduced CD8 T cells was determined by

flow cytometry on Day 5. Fold expansion was calculated over media control. Data points represent the mean of technical duplicates of E7 TCR-T

cells from 3 donors; error bars represent standard deviation; One-way ANOVA compared to RCT-CTRL, *P< 0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

4-1BBL = 4-1BB ligand; CTRL = control; HLAA2 = human leukocyte antigen A2; HPV = HLA-A2-human papillomavirus E7 peptide; IL = interleukin;

RCT= Red Cell Therapeutic experimental construct; RTX-321 = RTX-HPV-4-1BBL-IL-12 product candidate; TCR = T cell receptor.

• While signal 1 alone is sufficient for activation, all 3 signals are required for robust expansion of CD8 T cells

• Signal 1+2 on RCT-HPV-4-1BBL do not significantly expand antigen-specific cells compared to the RCT control treatment

• RTX-321 preferentially expands HPV E7 antigen-specific CD8 T cells over non-specific CD8 T cells

• Control RCT-CMV-4-1BBL-IL-12 does not expand HPV antigen-specific T Cells (E7 TCR-T) or non-HPV antigen-specific T cells (untransduced), indicating the requirement of cognate signal 1

Figure 3: Expression Profile of RTX-321 and Cognate HPV E7 TCR T Cells

(A) RTX-321 expression was determined by anti-IL-12, anti-41BBL, and anti-ββ2M staining. (B) CD8+ T cells from HLA-A*02:01 human donors were

transduced with lentivirus to express HPV E7-specific TCR with ~20-25% TCR expression (E7 TCR-T). (C) Untransduced CD8 T cells demonstrated

no transgenic or endogenous E7 TCR expression. E7 TCR expression was determined by HPV E7-specific tetramer and anti-mouse TCRβ β

antibody, detecting the transgenic mouse TCR beta chain.

HPV = human papillomavirus; RTX-321 = RTX-HPV-4-1BBL-IL-12 product candidate; TCR = T cell receptor.

RESULTS AND METHODS

Figure 4: RTX-321 Engages HPV-Specific T Cells

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 were incubated with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12, RCT-4-1BBL-IL-12, RCT-HPV-4-1BBL, RCT-CMV-4-1BBL-IL-12, or

RTX-321. (A) At 2 hours, Nur77+ percent and (B) at 24 hours, CD69+ percent of CD8 T cells were determined by flow cytometry to evaluate TCR

activation. Data points represent the mean of technical duplicates of E7 TCR-T cells from 3 donors; error bars represent standard deviation;

One-way ANOVA compared to RCT-CTRL, *P< 0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

4-1BBL = 4-1BB ligand; CD69 = cluster of differentiation 69; CTRL = control; HLAA2 = human leukocyte antigen A2; HPV = HLA-A2-human

papillomavirus E7 peptide; IL = interleukin; Nur77 = nuclear hormone receptor 77; RCT= Red Cell Therapeutic experimental construct;

RTX-321 = RTX-HPV-4-1BBL-IL-12 product candidate; TCR = T cell receptor.

• Presentation of HPV-signal 1 on MHC I engages antigen-specific TCR and induces T-cell signaling as determined by Nur77 upregulation on HPV E7 antigen–specific cells

• Presentation of HPV-signal 1 on MHC I induces early activation by upregulation of CD69 on HPV E7 antigen–specific cells

• Presentation of control RCT-CMV-4-1BBL-IL-12 does not induce T cell signaling and early activation on antigen-specific cells, elucidating the importance of cognate signal 1

Figure 7: RTX-321 Leads to the Accumulation of Central and Effector Memory Cells

E7 TCR-T cells were incubated with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12, RCT-4-1BBL-IL-12, RCT-HPV-4-1BBL, RCT-CMV-4-1BBL-IL-12,

or RTX-321. On Day 5, number of (A) Tcm and (B) Tem of E7 TCR-T CD8 T cells was determined by flow cytomery. Tcm was defined as

CD45RO+CCR7+ and Tem was defined as CD45RO+CCR7-. Data points represent the mean of technical duplicates of E7 TCR-T cells from 3

donors; error bars represent standard deviation; One-way ANOVA compared to RCT-CTRL, *P< 0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

4-1BBL = 4-1BB ligand; CTRL = control; HLAA2 = human leukocyte antigen A2; HPV = HLA-A2-human papillomavirus E7 peptide; IL = interleukin;

RCT= Red Cell Therapeutic experimental construct; RTX-321 = RTX-HPV-4-1BBL-IL-12 product candidate; Tcm = central memory T cell;

TCR = T cell receptor; Tem = effector memory T cell

• Signal 1+2 on RCT-HPV-4-1BBL drives memory differentiation of HPV E7 antigen-specific cells

• RTX-321 significantly enhances effector memory phenotype of HPV E7 antigen-specific cells

Figure 8: RTX-321 Induces Effector Molecules

E7 TCR-T cells were incubated with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12, RCT-4-1BBL-IL-12, RCT-HPV-4-1BBL, RCT-CMV-4-1BBL-IL-12,

or RTX-321. On Day 1, (A) Granzyme B+ percent of E7 TCR-T CD8 T cells was determined by flow cytomery. On Day 5, (B) IFNγβ secretion into the

supernatant was determined by Cytometric Bead Array. Data points represent the mean of technical duplicates of E7 TCR-T cells from 3 donors;

error bars represent standard deviation; One-way ANOVA compared to RCT-CTRL, *P< 0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

4-1BBL = 4-1BB ligand; CTRL = control; HLAA2 = human leukocyte antigen A2; GZMB = Granzyme B; HPV = HLA-A2-human papillomavirus E7

peptide; IL = interleukin; INFγβ = Interferon; RCT= Red Cell Therapeutics experimental construct; RTX-321 = RTX-HPV-4-1BBL-IL-12 product

candidate; TCR = T cell receptor.

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

CONCLUSIONS AND FUTURE PLANS

• Presentation of HPV on MHC I engages the TCR and initiates T-cell signaling and early activation on HPV antigen–specific cells

• RTX-321 induces activation determined by upregulation of 4-1BB, CD25, and PD-1 expression in vitro

• All 3 signals including antigen-specific signal 1 are required for robust expansion, effector memory phenotype, and effector molecule production of HPV antigen–specific cells

• RTX-321 induces T-cell mediated responses in an antigen-specific manner in vitro

• A Phase 1 clinical study is planned to evaluate RTX-321 for the treatment of patients with HPV 16+ cancers, including cervical and head and neck cancer

DISCLOSURES

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

RED PLATFORM®

ONE �HEALTHY�O- DONOR

EXPANSION & �DIFFERENTIATION

PROGENITOR �CELL COLLECTION

LENTIVIRAL VECTORENCODING OF MHC I

(HPV PEPTIDE), �CO-STIMULATORY

MOLECULE & CYTOKINE

ENUCLEATION & MATURATION

100-1000’s �OF DOSES

RED CELL THERAPEUTIC

RESULTS AND METHODS CONT'D

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mTCR+E7TET+23.1

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PD-1 %

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RTX-321

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E7 TCR-T

Granzyme B %

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