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 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.

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-2Db (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-2Kb (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 1×105 B16-F10 tumor cells on Day 0 followed by transfer of 2×106 naïve pmel-1 T cells on

Day 1. Mice (n=5 to 8) were then dosed with 1×109 mRBC-CTRL, 1×109 mRBC-CTRL with βαPD-1, or 1×109, 2.5×108, or 6×107 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 1×109 mRBC-CTRL, 1×109

mRBC-CTRL+βαPD-1, or 1×109 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; βPD-1=anti-programmed cell death protein 1; CTRL=control; D=Day;

gp100=H-2Db-gp100; IFNγβ=interferon γβ; 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 2×106 EG7.OVA cells and randomized when tumors reached ~230 mm3 (n=8),

treated with 1×106 naïve OT-1 cells, and dosed with 1×109 mRBC-CTRL or a dose titration of mRBC-OVA-4-1BBL-IL-12 (1×109, 2.5×108).

(B) Tumor growth curve after randomization and treatments. (C) In a separate study, CD45.1 Pep Boy mice were inoculated subcutaneously

with 2×106 EG7.OVA cells and randomized when tumors reached ~175 mm3 (n=5), treated with 1×106 naïve OT-1 cells, and dosed with 1×109

mRBC-CTRL or a dose titration of mRBC-OVA-4-1BBL-IL-12 (1×109, 2.5×108). (D) OT-1 numbers per gram of tumor on Day 7. (E) In a separate

study, C57BL/6 mice were inoculated subcutaneously with 2×106 EG7.OVA cells. Subsequently, 1×109 mRBC-CTRL, or a dose titration of

mRBC-OVA-4-1BBL-IL-12 (1×109, 3×108) was administered (n=8) on Days 1, 4, 8, and 11. (F) Tumor growth curves after randomization and

treatments. (G) Representative flow plots (1×109 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-2Kb-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-SpecificT Cell

T CellNK Cell

Signal 3 Cytokine: IL-12

Signal 2 Co-StimulatoryAgonist: 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 2×106 EG7.OVA cells. When the tumors reached a volume of ~175 mm3, the

animals were randomized (n=5) and treated with 1×106 naïve OT-1 cells. After, 1×109 mRBC-CTRL or a dose titration of mRBC-OVA-4-1BBL-

IL-12 (1×109, 2.5×108) was administered on Days 0 and 3. Mice were sacrificed on Day 7. (B) Polyfunctionality (granzymeB+IFNγβ+ %) in the

tumor-infiltration OT-1 cells on Day 7. (C) Ki67, TNFα β, and IL-2 per cell expression in tumor infiltrating endogenous CD8+ T cells; (D) Treg%

and IFNγβ+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 γβ=interferon γβ; 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-2Kb-OVA; Th1=T helper 1 CD4+ T cells; TNFα β=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 2×106 EG7.OVA cells. Subsequently, 1×109 mRBC-CTRL, or a dose titration of

mRBC-OVA-4-1BBL-IL-12 (1×109, 3×108) was administered (n=8) on Days 1, 4, 8, and 11 (noted with arrows in B-E). (B) Ki67+%, TEM %, and

granzymeB+% in CD8+ T cells; (C) Ki67+% in NK cells; (D) MHC-II+% of monocytes in the blood over time. (E) Plasma IFNγ β 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; IFNγβ=interferon γβ; IL=interleukin; MHC-II=major

histocompatibility complex class II; mRBC=murine red blood cell; NK=natural killer cells; OVA=H-2Kb-OVA; TEM=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 mm3 (n=8), treated with 1×106 naïve OT-1 cells, and dosed

with 2.5×108 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 5×105 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) TCRβ

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 TCRβ 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-2Kb-OVA;

TCRseq=TCRβ 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 1×106 naïve OT-1 transfer were dosed with 1×109 mRBC-CTRL or a dose titration of mRBC-OVA-

4-1BBL-IL-12 (1×109, 3×108) on Days 0, 4, 7 and 11 (noted with arrows in B and C). (B) Body weight changes compared to Day 0. (C) Plasma IFNγβ

levels over time. (D) Serum ALT levels on Days 12 and 25. (E) C57BL/6 mice (n=5) were dosed with 1×109 CellTraceTM 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; IFNγβ=interferon γ;

IL=interleukin; LN=lymph node; mRBC=murine red blood cell; OVA=H-2Kb-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

Met

asta

sis

Co

unt

Lung Metastasis Count

****

********

mRBC-gp100-4-1BBL-IL-12mRBC-CTRL

mRBC-CTRL+PD-1

6 107

2.510

8 1 10

9

mRBC-CTRL

mRBC-CTRL+PD-1

6 107

2.510

8 1 10

90

2 102

4 102

6 102

1 1042 1043 1044 1045 104

CD

90.1

+ CD

8+

Nu

mb

erin

50

µl B

loo

d Day 4

Pmel-1 Number (Blood)

Day 7 Day 11

mRBC-gp100-4-BBL-IL-12

****

****

**

Pmel-1 Number (Spleen)

0

1 105

2 105

3 105

4 105

CD

90.1

+ CD

8+

Nu

mb

er ****

mRBC-gp100-4-1BBL-IL-12 mRBC-gp100-4-1BBL-IL-12

0.0

5.0 104

1.0 105

1.5 105Pmel-1 Number (Lung)

CD

90.1

+ CD

8+ N

um

ber

****

0

20

40

60

80

100CD8 Functionality (Lung)

Eff

ecto

r+%

of C

D8

T C

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IFN +%

granzymeB+%

IFN +granzymeB+%

****

********

********

****

Dose

aAPC

T cell

1 10 9 1 10 9 2.5 10 86 10 7 1 10 9

+ Ctrl + + +

pmel-1 Endogenous

1 10 9

CtrlαPD-1 - - - - -+mRBC-CTRL

mRBC-CTRL+PD-1

6 107

2.510

8 1 10

9

mRBC-CTRL

mRBC-CTRL+PD-1

6 107

2.510

8 1 10

9

B

mRBC-CTRL

mRBC-CTRL and αPD-1

A

D0 D1 D4 D8 D14

B16-F101x105

Analyses

2x106

pmel-1

aAPC or αPD-1 Dosing

mRBC-gp100-4-1BBL-IL-12

OT-1 T Cell

EG7.OVATumor Cell

IL-12

mRBC-OVA-4-1BBL-IL-12

4-1BBL

OVA peptide

TCR

H-2Kb

pmel-1 T Cell

B16-F10Tumor Cell

IL-12

mRBC-gp100-4-1BBL-IL-12

4-1BBL

gp100 peptide

TCR

H-2Db

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

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 (8×105,

2×105, 5×104 and/or 1.25×104) were incubated with 2×105 PMBCs alone or 2×105 PBMC+ 4×103 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 β 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; IFNγβ=interferon γβ; 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

1Chunqing 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.

2Saraiya 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.

3Ndiaye 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

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CD8+ T Cells in Tumor

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1x109 mRBC-CTRL 1x109 mRBC-OVA-4-1BBL-IL-123x108 mRBC-OVA-4-1BBL-IL-12

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Tumor Volume mRBC-CTRL mRBC-OVA-4-1BBL-IL-12

Days After Tumor Randomization

Tu

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(mm

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OV

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D0 D1 D4 D8 D15

aAPC dosing

Analyses

D11

D-6 D1 D4

EG7.OVAaAPC dosing

D7

2x106

EG7.OVA2x106

1x106 OT-1

0

2 104

4 104

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1 105

**

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Days After Randomization

Tum

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Proliferating Th1% in Tumor

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D-8 D0 D3

Analyses

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2x106

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1x106 OT-1

Gra

nzy

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B+I

FNγ+

%

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Ki67 CD8 in Tumor

***

TNFα CD8 in Tumor

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*

IL-2 CD8 in Tumor

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1x109 mRBC-CTRL1x109

2.5x108

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Treg% CD4 in Tumor

**

1x109 mRBC-CTRL1x109

2.5x108

1x109 mRBC-CTRL1x109

2.5x108

1x109 mRBC-CTRL1x109

2.5x108

1x109 mRBC-CTRL1x109

2.5x108

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-121x109 mRBC-CTRL

1x109

2.5x108

1x109 mRBC-CTRL1x109

2.5x108

1x109 mRBC-CTRL1x109

2.5x108

1x109 mRBC-CTRL1x109

2.5x108

0

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Ki67 NK in Tumor

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M1 Macrophage % in Tumor

MH

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7 M

FI o

f CD

8+C

D45

.1+C

D45

.2-

TN

Fα

MF

I of C

D8

+CD

45.1

+CD

45.2

-

IL-2

MFI

of C

D8

+CD

45.1

+CD

45.2

-

E F G

1x109 mRBC-CTRL

1x109 mRBC-OVA-4-1BBL-IL-122.5x108 mRBC-OVA-4-1BBL-IL-12

CD8 T Cell Response

An

tig

en

-Sp

eci

fic R

esp

on

seA

dap

tive

Imm

un

e R

esp

on

se

Inn

ate

Imm

un

e R

esp

on

seA

dap

tive

Imm

un

e R

esp

on

se

CD4 T Cell Response NK Cell Response Macrophage Response

BA

0 5 10 150

20

40

60

80

100

% Ki67 CD8+ T Cells

Days

% K

i67+

/C

D8

+

********

** ***

***

**** *

0 5 10 150

20

40

60

80

% TEM CD8+ T Cells

Days

% C

D44

+/C

D6

2Lne

g/

CD

8+

********

*****

**

0 5 10 150

20

40

60

80

% GranzymeB CD8+ T Cells

Days

% g

ranz

ymeB

+/C

D8

+ ********

**

**

***

D %MHC-II+ of Monocyte

0 5 10 150

10

20

30

40

Days

%IA

-IE

+/C

D11

b+ ****

***

** ***

****

*

** ***

0 5 10 150

5000

10000

15000

Soluble 4-1BB

Days

pg

/m

L

**

**** *

**

*

C

0 5 10 150

50

100

% Ki67 of NK Cells

Days

% K

i67+

/N

K1.

1+/

CD

3ne

g **** **** ******

********

********

0 5 10 150

500

1000

1500

2000

IFN

Days

pg

/m

L

***

***

**

**

****

E

D0 D1 D4 D8 D15

EG7.OVA aAPC dosing

Analyses

D11

2x106

Innate Immune Response Soluble Factors

CD8 T Cell Response

1x109 mRBC-CTRL

1x109 mRBC-OVA-4-1BBL-IL-122.5x108 mRBC-OVA-4-1BBL-IL-12

A2x106

EG7.OVA

2x106

EG7.OVA

Days

01

4

7

646566707376

126127

136

1x105 EL4

OT-1

aAPC

TCRseqTetramer

TCRseq

Randomization

GF OT-1 Frequency in Blood

Day 65 Day 73 Day 126 Day 1360.0

0.1

0.2

0.3

0.4

0.5

CA

SS

RA

NY

EQ

YF

Fre

qu

ency

Treatment Naïve(No EL4 Inoculation)

mRBC-OVA-4-1BBL-IL-12 Cured

EG7.OVARechallenge

EL4Challenge

EL4 Challege

Tum

or

Vo

lum

e (m

m3 )

0 10 20 300

2000

4000

6000

Days Post EL4 Challenge1x105

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 TetramerCD

45.2

=OT

-1

B EG7.OVA Rechallenge

OT-1 (5x105)

Naive Group

0 10 20 30 400

2000

4000

6000

Days Post EG7.OVARechallenge

Tum

or

Vo

lum

e (m

m3 )

2x106

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

CD

45.2

+tet

ram

er+n

um

ber

Day 64 Day 70 Day 760

1000

2000

3000

Treatment NaïvemRBC-OVA-4-1BBL-IL-12 Cured

ND

****

E Endogenous OVA-Specific in Blood

Day 64 Day 70 Day 760

500

1000

1500

2000

CD

45.2

-te

tram

er+

num

ber

***

ND

H Sum Frequency of EL4 Expanded Clones

Day 65 Day 73 Day 126 Day 13610-5

10-4

10-3

10-2

10-1

Log

(Clo

ne F

req

uen

cy)

Non-Responder (1/7)Partial Responder (3/7)

EG7.OVA Rechallenge

EL4 Challenge

Complete Responder (3/7)

BA

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/mm2)

Density as a % of Spleen

DC

E F G

D1 D41h D8

aAPC DosingAnalyses

D0 D4 D11D7D12 D25

OT-1

aAPC Dosing Analyses1x106

1x109 mRBC-CTRL+OT-11x109 mRBC-OVA-4-1BBL-IL-12+OT-13x108 mRBC-OVA-4-1BBL-IL-12+OT-1

1x109 mRBC-CTRL1x109 mRBC-OVA-4-1BBL-IL-123x108 mRBC-OVA-4-1BBL-IL-12

Body Weight

0 5 10 15 20 25-10

-5

0

5

10

Days Post Dose 1

Bo

dy

Wei

ght

Cha

nge

(%)

*

*

1x109 mRBC-CTRL3x10

81x109

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 24 260

1 103

2 103

3 103

4 103

5 103

Days Post Dose 1

pg

/m

l

IFN

********

******* *

*****

Spleen

Liver

Lung

Bone marro

w

Mese

nteric

LN

Mandib

ular L

N

Thymus

Adrenal g

land

Heart

Kidney

Brain

Stom

ach

Inte

stin

e

Musc

le

Test

is

0

3000

6000

9000

12000

Biodistribution

7.9 fold****

mRBC-CTRL 1 HourmRBC-OVA-4-1BBL-IL-12 1h

mRBC-CTRL Day 8mRBC-OVA-4-1BBL-IL-12 Day 8

*** **** **

1 Hour Day 80

100

200

300

400

500

Bone Marrow

****

1 Hour Day 80

20

40

60

Heart

mRBC-CTRL

mRBC-OVA-4-1BBL-IL-12**

1 Hour Day 80

100

200

300

400

Liver

mR

BC

/m

m2

mR

BC

/m

m2

mR

BC

/m

m2

mR

BC

/m

m2

***

BA DCHLA-DR+% of Live Total CD8

HLA

-DR

+% o

f liv

e to

tal C

D8

0

20

40

60

80

***

********

***

FE 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

CD

8+

Tet

ram

er+

Nu

mb

er

NK Number

CD

3-C

D56

+ N

um

ber

DNAM1+% in NK Cells

DN

AM

1+%

of C

D56

+ N

K

GranzymeB+% in NK Cells IFNγ Secretion

Gra

nzym

eB+%

of C

D56

+ N

K C

ells

IFNγ

pg

/m

l

0

10

20

30

40

**

********

Tb

et+%

of L

ive

To

tal C

D8

GranzymeB+% in CD8+ T Cells Tbet+% in CD8+ Cells

0

20

40

60

80

100* ** ***

Gra

nzym

eB+%

of L

ive

To

tal C

D8

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