A Phase 1 Study of RTX-321, an Engineered Red Blood Cell as an Artificial Antigen-Presenting Cell Expressing HLA-A*02 with the HPV-16 E7 Peptide and 4-1BB Ligand with Membrane-Bound IL-12 for the Treatment of HPV 16-Positive Cancers

Johanna C. Bendell1, Alexander Spira2, Ramez Eskander3, Ezra Cohen3, Geoffrey Kuesters4, Christopher Plescia4, Karen Campbell4, Tiffany Chen4, Omid Hamid5 1Sarah Cannon Research Institute/Tennessee Oncology, 2Virginia Cancer Specialists, 3University of California San Diego, 4Rubius Therapeutics, 5The Angeles Clinic and Research Institute, a Cedars Sinai Affiliate

American Society of Clinical Oncology Annual Meeting/June 4-8, 2021

Figure 4. Trial Design

TRANSLATIONAL MEDICINE SUMMARY

• Analysis One: Peripheral blood immunophenotyping of the immune response including the assessment of the activation status and expansion of the innate immune response (monocytes, NK cells) and the adaptive response (CD4+ T cells, CD4+ regulatory T cells, CD8+ T cells)

• Analysis Two: E7-specific CD8+ T cells by tetramer analysis of peripheral whole blood and ELISpot analysis of isolated, live peripheral blood mononuclear cells (PBMC)

• Analysis Three: Immunophenotyping by immunofluorescence of the tumor microenvironment in paired tumor biopsies

• Analysis Four: Characterization of the tumor microenvironment by Nanostring IO panel analysis in paired tumor biopsies

Figure 5. Tetramer Analysis Gating

• T cells transgenic for the E7 T cell receptor (TCR-T) were spiked into PBMC preparations and assessed by tetramer staining by flow cytometry

• The gating strategy is depicted and a low vs high number TCR-T were spiked into the PBMC preparation and assessed for tetramer positive cells

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;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.

4 Zhang X., Luo M., Dastagir S.R. et al. Engineered red blood cells as an off-the-shelf allogeneic anti-tumor therapeutic. Nat Commun. 12, 2637 (2021).

5 Zhang X. et al. An Engineered Allogeneic Artificial Antigen-Presenting Red Cell Therapeutic™, RTX-321, Promotes Antigen-Specific T Cell Expansion and

Anti-Tumor Activity. FOCIS 2020.

6 Luo M et al. RTX-321, an Allogeneic Red Blood Cell-Based Artificial Antigen-Presenting Cell, Expressing MHC I Peptide, 4-1BBL, and IL-12, Engages Primary

Human HPV-Specific T Cells, and Boosts Other General Immune Responses. AACR Tumor Immunology and Immunotherapy 2020.

7 Nixon MJ et al. In Vivo Efficacy and Pharmacodynamic Analysis of RTX-321, an Engineered Allogeneic Artificial Antigen Presenting Red Cell Therapeutic.

AACR 2020.

• Published preclinical studies demonstrate that the mouse surrogate of RTX-321 promotes tumor control, memory formation and epitope spreading in tumor models in vivo4-7

• 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 spleen4-7

• Taken together, these findings support the potential of RTX-321 as an effective therapy for the treatment of HPV 16+ cancers

Figure 2. RTX-321 is a Cellular Therapy Expressing Three Cell Surface Proteins to Modulate the Immune System

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 ββ2 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.

Figure 3. RTX-321 Mechanism of Action

Preclinical studies demonstrate the temporal order of the pleiotropic effects of RTX-321 in the stimulation of an anti-tumor immune response. Following

infusion, innate immune activation is observed first (1) with increased MHC class II+ monocytes and NK cell proliferation. Following innate activation,

proliferation and activation of antigen-specific T cells is observed (2). NK and T cell mediate tumor killing, leading to release of tumor antigens (3), which are

then taken up by antigen-presenting cells, leading to broad tumor antigen presentation (4) and the process of epitope spreading (5). Together, these effects led

to a broad and effective immune response against multiple tumor antigens in preclinical models (6).

PHASE 1 CLINICAL TRIAL DESIGN

• The primary objective of the Phase 1 portion of the RTX-321-01 study is to determine the safety and tolerability of RTX-321 as monotherapy and to identify the recommended phase 2 dose (RP2D) of RTX-321 in patients with metastatic HPV 16-associated cancers

• Secondary objectives include: – To assess the pharmacodynamic changes in immune cell populations in the peripheral blood, including innate responses (NK cells, monocytes) and adaptive responses (enhanced activation and proliferation of CD4+ Th1 cells and E7-antigen specific CD8+ T cells)

– To determine the anti-tumor activity of RTX-321 as measured by ORR, duration of response, progression-free survival and overall survival

– To assess the pharmacokinetic effects of RTX-321 following intravenous dosing

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 immune biology 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 and is currently being evaluated in a Phase 1 clinical trial.

METHODS

Methods: The RTX-321-01 study is a Phase 1 multi-center, dose-escalation study of RTX-321 administered intravenously every 3 weeks in HLA-A*02:01-positive patients with relapsed or refractory HPV 16-positive cancers of the cervix or anal canal, or squamous cell cancers of the head and neck (HNSCC). Patients with cervical cancer or HNSCC will undergo testing for the presence of the HPV 16 virus or provide confirmation from archival tumor tissue prior to enrollment. Patients with anal cancer will not be required to have prospective determination of HPV 16-positive status prior to enrollment given the high incidence in this indication (approximately 80%-85% of anal cancers).

Patients will be enrolled across multiple dose-level cohorts to identify the recommended Phase 2 dose (RP2D) of RTX-321, followed by RP2D expansion cohorts in specific indications. The starting dose is 1 billion (1x109) cells administered intravenously every 3 weeks (Q3W) and the dose will escalate by half-log increments, following a Bayesian logarithmic regression model (BLRM) with overdose control. Translational studies will investigate the activation and expansion of HPV 16 E7 antigen-specific responses as well as broad innate and adaptive responses in multiple peripheral blood samples over the first 3 cycles of therapy as well as in optional paired tumor biopsies. At this time, the study is open and enrolling patients in the first dose escalation cohort (NCT04672980).

Figure 1. The RED PLATFORM® is Designed to Generate Allogeneic, Off-the-Shelf Cellular Therapies

MHC=major histocompatibility complex.

• The manufacturing process for Red Cell Therapeutics begins with early progenitor cells collected via leukapheresis from healthy O-negative and Kell-negative donors

• These early progenitor cells are then genetically engineered with a lentiviral vector to express hundreds of thousands of biotherapeutic proteins on the cell surface

• The cells are then exposed to media in a bioreactor to promote further expansion and differentiation until the nucleus is ejected, resulting in a mature reticulocyte with the biotherapeutic proteins on the cell surface

• This universal, scalable and consistent manufacturing process is being used in our fully owned manufacturing facility in Smithfield, RI, to supply our ongoing clinical trials

PRECLINICAL OBSERVATIONS

• RTX-321 and a murine surrogate model (mRBC-321) have demonstrated in vitro and in vivo, respectively, a dual mechanism of action (1) functioning as an aAPC to boost antigen-specific CD8+ T cell responses, and (2) promoting other target-antigen-independent stimulation of both innate and adaptive immune response

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

INTRODUCTION

Poster #TPS2664

Signal 1 Tumor Antigen: HPV 16 E7

RTX-321

MHC I (HLA-A2)

Antigen-SpecificT Cell

T CellNK Cell

Signal 3 Cytokine: IL-12

Signal 2 Co-StimulatoryAgonist: 4-1BBL