poster #p233 rtx-321, an allogeneic artificial antigen presenting cell (aapc… · 2020. 8. 3. ·...

RTX-321, an Allogeneic Artificial Antigen Presenting Cell (aAPC) Red Cell Therapeutic, Expressing MHC I-Peptide, 4-1BBL and IL-12, Promotes Expansion

and Anti-Tumor Activity of Antigen-Specific T Cells in HPV16-Positive TumorsXuqing Zhang, Mengyao Luo, Shamael R. Dastagir, Mellissa Nixon, Albert Lee, Naren Subbiah, Douglas C. McLaughlin, Chris Moore, Sneha Pawar, Nicholas Bayhi, Timothy J. Lyford, Omkar Bhate, Viral Amin, Christopher L. Carpenter, Thomas J. Wickham, and Tiffany F. Chen

Rubius Therapeutics, Cambridge, MA, USA

Poster #P233

Figure 10. RTX-321 (HPV-4-1BBL-IL-12) is Active in HPV-Specific TCR Transduced T Cell Line and Primary Human T Cells

(A) RTX-321 expression was determined by anti-IL-12, anti-4-1BBL and anti-ββ2M staining. (B) 4×105 Jurkat cells with a NFAT luciferase reporter engineered to express HPV E7-specific TCR were incubated with RCT-CTRL, RCT-HPV, RCT-4-1BBL, RCT-IL-12 or RTX-321 at a range of doses (3.2×106, 1.6×106, 8×105, 4×105, 2×105, 1×105). After 18- to 22-hour incubation, a luciferase reporter assay was carried out to determine NFAT signal-fold change compared to Jurkat reporter cell alone. (C) 3.5×104 4-1BB/NFβB reporter HEK293 cell line that expresses human 4-1BB and has a NFκβB luciferase reporter construct stably integrated was incubated at a range of doses of RTX-321 and controls (4×105, 2×105, 1×105, 5×104, 2.5×104, 1.25×104, or 6.25×103). After 18 to 22-hour incubation, a luciferase reporter assay was carried out to determine NFβκβB signal fold-change compared to reporter cell alone. (D) 5×104 HEK-Blue IL-12 cells, expressing a STAT4-inducible SEAP reporter gene and the IL-12 receptors, were incubated with RTX-321 and controls at different doses (5×105, 1.7×105, 5.6×104, 1.9×104, 6.2×103, or 2.1×103) for 24 hours. SEAP expression was measured by absorbance at 655 nm and shown as fold-change relative to the reporter cell alone control. (E) CD8+ T cells from a human donor were transduced with lentivirus to express an HPV E7-specific TCR. 8×104 TCR engineered primary CD8+ T cells with ~31% TCR expression, and untransduced T cells were incubated with 1.6×105 RTX-321 and controls for 7 days. HPV E7 tetramer+ CD8 T cell numbers were determined by flow cytometry.

4-1BBL = 4-1BB ligand; β2m = beta-2-macroglobulin; CTRL = control; HPV = HLAA2-human papilloma virus peptide; IL = interleukin; NFAT = nuclear factor of activated T cells; NFκββB = nuclear factor kappa-light-chain-enhancer of activated B cells; RCT = Red Cell Therapeutic experimental construct; RTX = Red Cell Therapeutic product candidate; RTX-321 = red cell therapeutic-HPV-4-1BBL-IL-12; SEAP = Secreted embryonic alkaline phosphatase; SSC = side scatter; TCR = T cell receptor; UNT = untransduced control; HLAA2 = human leukocyte antigen A2.

• RTX-321 selectively directs against HPV dominant epitope in HPV+ cancer patients and robustly engages TCR, 4-1BB and IL-12R signaling in engineered cell lines

• RTX-321 expands HPV-specific TCR-transduced primary human T cells

CONCLUSIONS• RTX-aAPCs activate and significantly expand antigen-specific T cells, reduce tumor burden and have minimal,

reversible toxicity

• Expression of signal 3 on an RTX-aAPC results in robust antigen-specific T cell expansion, memory formation and cytotoxicity towards tumor cells

• RTX-aAPC targeted against a tumor-associated antigen, gp100, promotes antigen-specific T cell expansion and effector function, and nearly eliminates B16-F10 lung metastases at the highest doses

• RTX-aAPC targeting CMV epitope expands CMV-specific T cells from CMV+ healthy donor PBMC

• RTX-321 (HPV-4-1BBL-IL-12) activates an HPV-specific TCR transduced T cell line and primary human T cells

• Clinical studies are planned to evaluate RTX-321 for the treatment of patients with HPV 16+ cancers, including cervical and head and neck cancer

ACKNOWLEDGEMENTSPoster design support was provided by Dennig Marketing Group, sponsored by Rubius Therapeutics. We would like to thank Lori Melançon for her editorial contributions.

DISCLOSURESAll authors: Employment with and equity ownership in Rubius Therapeutics.

RESULTS AND METHODS

Figure 5. IL-12 as Signal 3 on aAPC Significantly Enhances Efficacy and Potency in a Subcutaneous EG7.OVA Tumor Model

(A) CD45.1 Pep Boy mice were injected subcutaneously with 2×106 EG7.OVA cells. When the tumors reached a volume of approximately 150 mm3, the animals were randomized and dosed on Day 1 post randomization with 1×106 naïve OT1 cells. The animals were then dosed on Days 1, 4 and 7 with 2.5×108 of mRBC-CTRL, mRBC-OVA-4-1BBL, mRBC-OVA-4-1BBL-IL-7, mRBC-OVA-4-1BBL-IL-12 or mRBC-OVA-4-1BBL-IL-15. An additional control group received 200 μL PBS. (B) Tumor volumes were monitored every 2-3 days. (C) Survival and (D) tumor volume and regression were monitored in 1×109 mRBC-CTRL or mRBC-OVA-4-1BBL-dosed mice as well as 4-fold lower (2.5×108) and 16-fold lower (6.25×107) mRBC-OVA-4-1BBL-IL-12-dosed mice.

4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; IL = interleukin; mRBC = murine red blood cell; OVA = H2Kb-ovalbumin; PBS = phosphate buffered saline.

• mRBC-OVA-4-1BBL-IL-12 demonstrates superior tumor control efficacy compared to mRBC-OVA-4-1BBL, mRBC-OVA-4-1BBL-IL-7 or mRBC-OVA-4-1BBL-IL-15. Based on these results, IL-12 is chosen as signal 3 for RTX-aAPC

• mRBC-OVA-4-1BBL-IL-12 significantly increases survival

• A significant number of tumor regressions were observed at the 2.5×108 and 6.25×107 doses of mRBC-OVA-4-1BBL-IL-12

Figure 6. mRBC-OVA-4-1BBL-IL-12 Treatment Results in Minimal, Reversible Toxicity In Vivo, Likely Due to Restriction to the Vasculature

(A) Wildtype mice were dosed with 1×109 mRBC-CTRL or 1×109, 3×108, or 6×107 mRBC-OVA-4-1BBL-IL-12 on Days 0, 4, 7 and 11. Mice were sacrificed on Day 12 or on Day 25. (B) Body weight changes compared to Day 0 were monitored overtime. (C) IFNγ level in plasm was determined by cytokine bead array overtime. (D) Serum alanine aminotransferase (ALT) levels on Days 12 and 25 were determined by clinical chemistry.

4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; ALT = alanine aminotransferase; CTRL = control; IFNγ = interferon γ; IL = interleukin; mRBC = murine red blood cell; OVA = H2Kb-ovalbumin.

• No significant body weight changes were observed

• Serum IFNγ level returned to baseline after a recovery period

• Elevated serum alanine aminotransferase returned to baseline after a recovery period

Figure 7. mRBC-OVA-4-1BBL-IL-12 Expands Endogenous OVA-Specific T cells

CD45.1 Pep Boy mice were transferred with 2×106 naïve OT1 cells on Day 0 followed by dosing with 1×109 mRBC-CTRL or 1×109, 3×108, or 6×107 mRBC-OVA-4-1BBL-IL-12 on Days 0, 4, 7 and 11. Mice were sacrificed on Day 12 to determine OT1 and OVA-tetramer+ T cell number in (B) 30 μL blood and (C) spleen.

4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; IL = interleukin; mRBC = murine red blood cell; OVA = H2Kb-ovalbumin.

• mRBC-OVA-4-1BBL-IL-12 drives expansion of endogenous OVA-specific T cells. The expansion of the endogenous OVA-specific T cells is comparable to that of the OT1 cells one day after last dose in circulation and the spleen

Figure 3. The Addition of IL-7, IL-12 or IL-15 as Signal 3 on an RTX-aAPC Further Promotes Antigen-Specific T cell Expansion, Memory Formation and Cytotoxicity Towards Tumor Cells

1×105 OT1 T cells were incubated with 1×106, 3.3×105 or 1×105 mRBC-CTRL, mRBC-OVA-4-1BBL, mRBC-OVA-4-1BBL-IL-7, mRBC-OVA-4-1BBL-IL-12 or mRBC-OVA-4-1BBL-IL-15 at 37°C for 4 days. (A) OT1 CD8+ T cells were cultured with RCT-OVA-4-1BBL, RCT-OVA-4-1BBL-IL-7, RCT-OVA-4-1BBL-IL-12 or RCT-OVA-4-1BBL-IL-15 at 1:10 ratio. OT1 cells were harvested after a 3-day incubation and treated with ACK buffer to lyse the RTX-aAPC. 1×104 tumor cells, either parental tumor cells, EL4, or tumor cells expressing ovalbumin, EG7.OVA, were then incubated with expanded OT1 cells (effector cells) at 5:1, 2:1, 1:1, 0.5:1 and 0:1 effector to target (E:T) ratio. After a 22-hour incubation, cells were stained with live/dead dye and fixed with 2% paraformaldehyde to enumerate live target cells in each well. (B) OT1 numbers, (C) Tscm (CD122+CD62L+CD44-) OT1 number, (D) Tcm (CD122+CD62L+CD44+) OT1 number, and (E) Tem (CD122+CD62L-CD44+) OT1 number were quantified by flow cytometry. (F) EL4 percent killing and (G) EL7.OVA percent killing were calculated by live tumor cell percent of target only controls.

4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; IL = interleukin; MHC = major histocompatibility complex; mRBC = murine red blood cell; OVA = H2Kb-ovalbumin; RCT = Red Cell Therapeutic experimental construct; RTX-aAPC = Red Cell Therapeutic artificial antigen presenting cell; Tcm = central memory T cell; Tem = effector memory T cell; TCR = T cell receptor; Tscm = stem cell memory T cell.

• IL-7, IL-12 and IL-15 as signal 3 on a mouse (m) RBC-aAPC increases total antigen-specific T cell expansion and memory formation compared to mRBC-aAPC that expresses only signals 1 and 2

• IL-12 as signal 3 significantly improves antigen-specific killing of tumor cells compared to T cells expanded by RCT-OVA-4-1BBL, RCT-OVA-4-1BBL-IL-7 or RCT-OVA-4-1BBL-IL-15

Figure 4. IL-12 as Signal 3 on aAPC Promotes Antigen-Specific T Cell Expansion and Effector Function In Vivo

(A) CD45.1 Pep Boy mice were inoculated subcutaneously with 2×106 EG7.OVA cells. When the tumors reached a volume of approximately 180 mm3, the animals were randomized and treated on Day 0 with 1×106 naïve OT1 cells. 1×109 mRBC-CTRL, mRBC-OVA-4-1BBL, mRBC-OVA-4-1BBL-IL-7, mRBC-OVA-4-1BBL-IL-12 or mRBC-OVA-4-1BBL-IL-15 was administered on Days 0 and 3. A control group received 200 μL of PBS. (B) Total OT1 number (CD45.2+CD8+) and (C) Memory OT1 number (Tscm:CD122+CD62L+CD44-; Tcm: CD122+CD62L+CD44+; Tem: CD122+CD62L-CD44+) in 50 μL blood on days 0, 3 and 6 were determined by flow cytometry. (D) Mice were sacrificed on Day 6 to evaluate OT1 number in tumor, draining lymph nodes and spleen. (E) Genomic DNA was extracted from single cell suspension of the tumor and OT1 clone frequency within tumor infiltrating T cells was determined by TCRβ sequencing. (F) Memory OT1 numbers in the spleen on Day 6 were determined by flow cytometry. (G) Splenocytes were stimulated with PMA and ionomycin and OT1 effector function was determined by intracellular cytokine staining.

4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; dLN = draining lymph node; GzmB = granzyme B; IFNγ = interferon γ; IL = interleukin; mRBC = murine red blood cell; OVA = H2Kb-ovalbumin; Tcm = central memory T cell; Tem = effector memory T cell; Tscm = stem cell memory T cell.

• mRBC-OVA-4-1BBL-IL-12 and mRBC-OVA-4-1BBL-IL-15 induce dramatic expansion of circulating OT1 in vivo compared to mRBC-OVA-4-1BBL or mRBC-OVA-4-1BBL-IL-7

• Circulating OT1 cells expanded by mRBC-OVA-4-1BBL-IL-12 and mRBC-OVA-4-1BBL-IL-15 have a predominant effector phenotype while maintaining a central memory T cell population

• mRBC-OVA-4-1BBL-IL-12 induces >100-fold expansion of OT1 in the spleen compared to mRBC-CTRL

• Significantly more OT1 T cells are detected in the tumor of mRBC-OVA-4-1BBL-IL-12-dosed mice compared to mRBC-CTRL by TCR sequencing

• mRBC-OVA-4-1BBL-IL-12 maintained high level central memory T cell expansion in secondary lymphoid organs as seen with mRBC-OVA-4-1BBL

• mRBC-OVA-4-1BBL-IL-12 promotes antigen-specific T cell functionality (IFNγ and Granzyme B) as well as increases polyfunctional (IFNγ+ and Granzyme B+) T cells

INTRODUCTION• Red Cell Therapeutics™ (RCTs) are a new class of allogeneic, off-the-shelf cellular

therapeutic candidates for the treatment of cancer, rare diseases and autoimmune diseases

• For the treatment of cancer, allogeneic Red Cell Therapeutic artificial antigen presenting cells (RTX-aAPCs) are engineered to induce a tumor-specific immune response by expanding antigen-specific T cells. Rubius Therapeutics’ first artificial antigen-presenting cell product candidate, RTX-321, is for the potential treatment of HPV 16-positive cancers

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

• Delivered at a dose of <1% of total red blood cell volume in the body

• Universal, scalable and consistent manufacturing process

Figure 2. RTX-aAPC Drives Antigen-Specific Activation and Proliferation of T cells

MHC = major histocompatibility complex; RTX-aAPC = Red Cell Therapeutic artificial antigen-presenting cell; TCR = T cell receptor.

• RTX-aAPCs are engineered to simultaneously express on the cell surface a tumor-specific antigen on the MHC I, a co-stimulatory signal and a cytokine to mimic the human immunobiology of T cell-APC interactions

OBJECTIVES• To determine the optimal signal 3 on an RTX-aAPC to promote robust antigen-specific

T cell expansion, memory formation and cytotoxicity towards tumor cells

• To determine whether RTX-aAPCs have broad antigen applicability, using OVA, gp100 peptide and HPV antigens

Signal 3 cytokine

Signal 2 co-stimulatory agonist

Antigen-specific TCR

MHC I

Signal 1 Tumor antigen

RTX-aAPC

T CELLS

Figure 8. aAPC Targeted Against a Tumor-Associated Antigen, gp100, Promotes Antigen-Specific Pmel-1 T Cell Expansion, Effector Function and Dramatically Reduces B16-F10 Lung Metastases

(A) Wildtype 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 were then dosed with 1×109 mRBC-CTRL 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 1×109 mRBC-CTRL and 1×109 mRBC-gp100-4-1BBL-IL-12 dosed mice. (C) Lung metastasis counts in all groups were enumerated. Pmel-1 T cell number in (D) 50μL blood overtime, (E) the spleen, and (F) the left lobe of perfused lung were determined by flow cytometry. (G) Single-cell suspensions from perfused lung were stimulated with PMA and ionomycin followed by intracellular cytokine staining to determine effector function of lung infiltrating Pmel-1 and endogenous CD8+ T cells.

4-1BBL = 4-1BB ligand; aAPC = artificial antigen-presenting cell; CTRL = control; GzmB = granzyme B; gp100, H2Db-gp100; IFNγ = interferon γ; IL = interleukin; mRBC = murine red blood cell.

• mRBC-gp100-4-1BBL-IL-12 nearly eliminates lung metastases at the highest dose levels and dramatically reduces lung metastases at low dose levels in a B16-F10 melanoma model

• 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 9. RCT-CMV-4-1BBL-IL-12 Expands CMV-Specific T cells From CMV+ Healthy Donor PBMC

2×105 HLAA2+CMV+ human PBMC were incubated with 8×105 RCT-CTRL, RCT-CMV, RCT-CMV-4-1BBL or RCT-CMV-4-1BBL-IL-12 at 37ºC for 10 days. (A) CMV tetramer+ CD8 T cell numbers and (B) their memory phenotype were determined by flow cytometry. (C) IFNγ producing CMV-specific T cells numbers were quantified by ELISPOT post CMV peptide stimulation with representative images in (D).

4-1BBL = 4-1BB ligand; CMV = HLAA2-cytomegalovirus peptide; CTRL = control; IFNγ = interferon γ; IL = interleukin; PBMC = periphery blood mononuclear cell; RCT = Red Cell Therapeutic experimental construct; Tcm = central memory T cell; Tem = effector memory T cell; Temra = terminally differentiated effector memory; Tscm = stem cell memory T cell; TNTC = too numerous to count.

• RCT-CMV-4-1BBL-IL-12 expands CMV-specific T cells from healthy donor PBMC by both tetramer staining and ELISPOT measurements

• RCT-CMV-4-1BBL-IL-12 promotes memory generation within CMV tetramer+ population

Society for Immunotherapy of Cancer / Nov. 6-10, 2019 / National Harbor, ME

RED PLATFORM®

EXPANSION & �DIFFERENTIATION

PROGENITOR �CELL COLLECTION

ONE �HEALTHY�O- DONOR

ENUCLEATION & MATURATION

GENETIC �ENGINEERINGMHC-PEPTIDE

SIGNAL 2 AGONIST

100-1000’s �OF DOSES

RED CELL THERAPEUTIC

0 1 106 2 106 3 106 4 1060

2

4

6

8

10

TCR Activity

Cell Number

Fold

Ch

ang

e in

Lu

min

ese

nce

to H

PV

TC

R+

Jurk

at N

FAT

Re

po

rte

r

RCT-CTRL

RCT-IL-12

RCT-HPV

RCT-4-1BBL

RTX-321

β2m

4-1B

BL

IL-12

SS

C

RTX-321 4-1BBL Activity

0 1 105 2 105 3 105 4 105 5 1050

5

10

15

Cell Number

Fold

Ch

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Lu

min

esc

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r H

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RCT-IL-12

RCT-HPV

RTX-321

0 2 105 4 105 6 1050

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IL-12 Activity

Cell Number

Fold

Ch

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e in

SE

AP

to

Me

dia

Alo

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CT

RL

RCT-CTRL

RCT-IL-12

RCT-HPV

RCT-4-1BBL

RTX-321

RCT-CTRL

RCT-HPV-4-1BBL

RCT-4-1BBL-IL-12

RTX-321 0

200

400

2000

4000

6000

8000

10000

CD8+ HPV-E7 Tetramer + Number

Tota

l CD

8+ H

PV

-E7

Tetr

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r+ C

ou

nt

Day 7 UNT

Day 7 HLAA2-HPV TCR

A B C

D E

RCT-CTRL

RCT-CM

V

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V-4-1

BBL

RCT-CM

V-4-1

BBL-IL-1

20

5

10

15

20

Donor 1

CM

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r +

Co

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20

20

40

60

80

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Donor 2

CM

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Co

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20

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Donor 3

CM

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RCT-CTRL

RCT-CM

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V-4-1

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20

50

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Donor 3

% M

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ory

of

Tetr

ame

r+

Naive/Tscm (CD45RO-CCR7 +)Tcm (CD45RO +CCR7 +)Tem (CD45RO +CCR7 -)Temra (CD45RO-CCR7 -)

RCT-CTRL

RCT-CM

V

RCT-CM

V-4-1

BBL

RCT-CM

V-4-1

BBL-IL-1

20

100

200

300

400

500

Donor 1

Tota

l IFN

Sp

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/2×

105 P

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C

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20

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Donor 2

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2

0.0

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1.5 103

Donor 3

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TNTC

A B

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4 102

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Pmel-1 Number (Blood)

Days After Tumor Injection

Pm

el-

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Pm

el-

1 N

um

be

rs

in 5

0 µ

l Blo

od

1 109 mRBC-CTRL

1 109

2.5 108

6 107

****

Dose

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

mRBC-CTRL1 109

2.5 108

6 1070

1 105

2 105

3 105

4 105

Pmel-1 Number (Spleen)

**

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

1 109

2.5 108

6 1070

1 105

2 105

3 105

4 105

Pmel-1 Number (Spleen)

CD

90.1

+ CD

8+

Nu

mb

er **


0

20

40

60

80

100

CD8 Functionality (Lung)

Eff

ect

or+ %

of

CD

8 T

ce

lls

IFN +%

GzmB+%

IFN +GzmB+%

**

****

****

**

DoseaAPC

T cell

1 109 1 109 2.5 108 6 1071 109

+ Ctrl + + +

Pmel-1 endogenous

E F G

mRBC-CTRL


Take DownaAPC DosingB16-F10

Pmel-1D0 D1 D4 D8 D14

mRBC-CTRL1 109

2.5 108

6 107 0

50

100

150

200

250

Lung Metastasis Count

Lun

g M

eta

stas

is C

ou

nt

**

****


A C DB

D0 D4 D11

aAPC Dosing

D7

OT1

D12

Take Down

Antigen Specific T cells (Spleen)

1 109 mRBC-CTRL1 109

3 108 6 107

0.0

5.0 102

1.0 103

1.5 103

2.0 103

2.5 103

3.0 103

Antigen Specific T cells (Blood)

OV

A S

pe

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T c

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Nu

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in 3

0μl

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**

mRBC-OVA-4-1BBL-IL-121 109mRBC-CTRL

1 109

3 108

6 1070

2 103

4 103

6 103

8 103

1 104

OV

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pe

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T c

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

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*: p<0.05**: p<0.01compared to mRBC-CTRLstudent t-test

A CB

BA DC

1x10 9 mRBC-CTRL

1x10 9 mRBC-OVA-4-1BBL-IL-12

3x108 mRBC-OVA-4-1BBL-IL-12


1x10 9 mRBC-CTRL

1x10 9 mRBC-OVA-4-1BBL-IL-12



0 5 10 15 20 25-10

-5

0

5

10

Body Weight

Days Post Dose 1

Bo

dy

We

igh

t C

han

ge

(%)

Dose

IFN

Dose

Liver Enzymes (ALT)

1x109 mRBC-CTRL

1x109

3x108

6x1070

100

200

300

400

ALT

(U/

L)

*

Day 12

Day 25

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

0 2 4 6 8 10 12 14 16 18 20 22 24 260

500

1000

1500

2000

2500

3000

3500

Days Post Dose 1

IFN

(pg

/m

l)

Take DownaAPC Dosing

D0 D4 D7 D11D12 D25

mRBC-CTRL 0/8 regressionsmRBC-OVA-4-1BBL 2/8 regressions

mRBC-OVA-4-1BBL-IL-12 5/8 regressions; 16-fold lower dose

mRBC-OVA-4-1BBL-IL-12 7/8 regressions; 4-fold lower dose

PBS

mRBC-OVA-4-1BBL (2.5 10 8)

mRBC-OVA-4-1BBL-IL-12 (2.5 10 8)

mRBC-OVA-4-1BBL-IL-7 (2.5 10 8)

mRBC-OVA-4-1BBL-IL-15 (2.5 10 8)

0 5 10 15 20 250

1000

2000

3000

4000

5000

Days After Tumor Randomization

Tum

or

Vo

lum

e (m

m3 )

Tumor Volume

Dose

PBS

mRBC-CTRL

mRBC-OVA-4-1BBL-IL-12 (4-fold lower dose)

mRBC-OVA-4-1BBL-IL-12 (16-fold lower dose)

mRBC-OVA-4-1BBL

0 5 10 15 20 25 30 35 40 450

50

100

Survival


Pe

rce

nt

Su

rviv

al

p<0.0001

p<0.0001

Log rank test compared to mRBC-CTRL

Dose

D1 D4 D7

OT1aAPC dosing

D-7

EG7.OVA

0 5 10 15 20 250

1000

2000

3000


Tum

or

Vo

lum

e (m

m3 )

Efficacy Potency

Dose

C DBA

mRBC-CTRL

mRBC-OVA-4-1BBL-IL-120.0

0.2

0.4

0.6

0.8

1.0

OT1 Clone Frequency (Tumor)

CA

SS

RA

NY

EQ

YF

Clo

ne

% **

PBS

mRBC-CTRL

mRBC-OVA-4-1B

BLIL-15 IL-12 IL-7

0

2 105

4 105

6 105

8 105

OT1 Phenotype (Spleen)

OT

1 N

um

be

r

Tscm

Tcm

Tem

mRBC-OVA-4-1BBL

PBS

mRBC-CTRL

mRBC-OVA-4-1B

BLIL-15 IL-12 IL-7

0.0

5.0 105

1.0 106

1.5 106

OT1 Functionality (Spleen)

OT

1 N

um

be

r

IFN

mRBC-OVA-41BBL

GzmB

IFN +GzmB+

PBS

mRBC-CTRL

mRBC-OVA-4-1B

BLIL-15 IL-12 IL-7

04 102

1 104

2 104

5.0 104

1.0 105

1.5 105

OT1 Phenotype (Blood)

mRBC-OVA-4-1BBL

Tscm

Tcm

Tem

PBS

mRBC-CTRL

mRBC-OVA-4-1B

BLIL-15 IL-12 IL-7

0

1 105

2 105

3 105

5.0 105

1.0 106

1.5 106

OT1 Number

OT

1 N

um

be

r

mRBC-OVA-4-1BBL

Spleen

dLN

TumorD0 D3 D6

OT1 Take downaAPC dosing

D-8

EG7.OVA

PBS

mRBC-CTRL

mRBC-OVA-4-1B

BLIL-15 IL-12 IL-7

0.01.5 1033.0 103

5.0 104

1.0 105

1.5 105

OT1 Expansion (Blood)

OT

1 N

um

be

r in

50

µl B

loo

d

OT

1 N

um

be

r in

50

µl B

loo

d

Day 0

Day 3

Day 6

mRBC-OVA-4-1BBL

CBA D

E F G

B

0

1 10 4

2 10 4

3 10 4

4 10 4

OT

1 N

um

be

r

Total OT1 Number C

0.0

5.0 10 2

1.0 10 3

1.5 10 3

CD

122+

CD

44-C

D6

2L+

#

Tscm OT1 Number

E

0.0

5.0 10 3

1.0 10 4

1.5 10 4

CD

122+

CD

44+C

D6

2L+

#

Tcm OT1 Number

F

0.0

5.0 10 2

1.0 10 3

1.5 10 3

2.0 10 3

CD

122+

CD

44+C

D6

2L-

#

Tem OT1 Number

mRBC-CTRLmRBC-OVA-4-1BBL

mRBC-OVA-4-1BBL-IL-7mRBC-OVA-4-1BBL-IL-12mRBC-OVA-4-1BBL-IL-15

D

EL4

5:1 2:1 1:1 0.5:10

20

40

60

80

100

OT1:Target

% K

illin

g

G EG7.OVA

5:1 2:1 1:1 0.5:10

20

40

60

80

100

OT1:Target

% K

illin

g

RCT-OVA-4-1BBL

RCT-OVA-4-1BBL-IL-7

RCT-OVA-4-1BBL-IL-12

RCT-OVA-4-1BBL-IL-15

A

4-1BB-L

OVA specific TCRMHC I

OVA peptide

Signal 3 cytokine

Tumor Cell Killing

Activation/Expansion

OT1 T cell

Tumor cell

EG7.OVA EL4 (parental)

mRBC-aAPC (OVA) OT1 T cell

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