xuqing zhang, mengyao luo, shamael r dastagir, mellissa nixon, … · 2021. 6. 30. · xuqing...
TRANSCRIPT
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
ells
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
D7
2x106
EG7.OVA
1x106 OT-1
CBA
102
103
104
105
106
CD8+ T Cells in Tumor
Co
unt
s/G
ram
Tu
mo
r
****
******
39-fold
11-fold
Tetramer+ CD8 Tetramer- CD8
**
Tumor Volume
1 109 m
RBC-CTRL
2.510
8 1 10
9
CD
45.2
+tet
+CD
8+
Nu
mb
er/
Gra
m T
um
or
mRBC-OVA-4-1BBL-IL-12
E F G
1x109 mRBC-CTRL
1x109 mRBC-OVA-4-1BBL-IL-122.5x108 mRBC-OVA-4-1BBL-IL-12
1x109 mRBC-CTRL 1x109 mRBC-OVA-4-1BBL-IL-123x108 mRBC-OVA-4-1BBL-IL-12
0 5 10 15 200
500
1000
1500
2000
Tumor Volume mRBC-CTRL mRBC-OVA-4-1BBL-IL-12
Days After Tumor Randomization
Tu
mo
r V
olu
me
(mm
3 )
OV
A T
etr
ame
r** ***
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
6 104
8 104
1 105
**
0 5 10 15 20 250
500
1000
1500
2000
Days After Randomization
Tum
or V
olu
me
(mm
3 )
CD8
D
CBA
Proliferating Th1% in Tumor
aAPC dosing
D-8 D0 D3
Analyses
D7
2x106
EG7.OVA
1x106 OT-1
Gra
nzy
me
B+I
FNγ+
%
0
2000
4000
6000
8000
Ki67 CD8 in Tumor
***
TNFα CD8 in Tumor
0
5000
10000
15000
*
IL-2 CD8 in Tumor
0
200
400
600
***
1x109 mRBC-CTRL1x109
2.5x108
0
10
20
30
40
50
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
20
40
60
80
****
**
0
500
1000
1500
Ki67 NK in Tumor
Ki6
7 M
FI o
f NK
1.1+
CD
3-
*
GranzymeB NK in Tumor
Gra
nzy
me
B M
FI o
f NK
1.1+
CD
3-
0
500
1000
1500
2000
2500
**
0
10
20
30
40
M1 Macrophage % in Tumor
MH
CII+
F4/
80
+CD
11b
+Ly6
C-/
low
Ly6
G-C
D4-
CD
8-%
of C
D45
.1+
*
**
mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12 mRBC-OVA-4-1BBL-IL-12
Foxp
3+C
D25
+% o
f CD
4+C
D45
.1+
IFN
+K
i67+
% o
f CD
4+C
D45
.1+
Ki6
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