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Poster #144 RTX-240, an Allogeneic Engineered Red Blood Cell Expressing 4-1BBL and IL-15TP, Promotes NK Cell Functionality In Vitro and In Vivo
Anne-Sophie Dugast, Shannon L. McArdel, Enping Hong, Arjun Bollampalli, Maegan E. Hoover, Sneha Pawar, Viral Amin, Kangjian Qiao, Christopher Ta, Laurence A. Turka, Thomas J. Wickham, Sivan Elloul
Society of Immunotherapy of Cancer (SITC) 2020/November 9-14, 2020
Figure 5: RTX-240 Expands CD56dim NK Cells In Vitro
(A) Purified NK cells using negative selection (CD3/CD19 negatives CD56/CD16 positives) from 5 different donors were incubated for 8 days with RCT-CTRL, RTX-240, or with rhIL-15TP used as control.
NK cell phenotype was evaluated by flow cytometry, (B) shows representative flow cytometry plots of NK cells phenotype from one donor based on the expression of CD16 and CD56, and (C)
represents the counts of CD56dim NK cells for each treatment. Mean and standard deviation values are plotted across all 5 donors. One-way Anova compared to RCT-CTRL. **P≤0.01.
CD56=cluster of differentiation 56; NK cells=natural killer cells; IL=interleukin; RCT-CTRL=untransduced control; RTX-240=RTX-4-1BBL-IL-15TP product candidate; rIL-15=recombinant IL-15.
• RTX-240 expands CD56dim NK cells, a known population with cytotoxic activities, compared to rIL-15
Figure 6: RTX-240 Enhances NK Cell Cytotoxicity Against the Myeloid Leukemia Cell Line K562
(A) RTX-240 was incubated with purified NK cells using negative selection from 5 different donors at a 5:1 ratio overnight. On the next day, CellTrace™ Far Red (CTFR) dye-labeled K562 target cells were
added to the wells at a 5:1 target: NK cells ratio and incubated for an additional 4 hours. The percentage of degranulation (CD107a+) (B), and activation (CD69+) (C), of NK cells was evaluated by flow
cytometry, and (D) the percentage of dead K562 cells was determined by flow cytometry for each donor and determined by the percentage of LIVE/DEAD™ Fixable Aqua Dead Cell Stain staining of
total CTFR+ cells. Mann-Whitney compared to control. **P≤0.01.
CD69=cluster of differentiation 69; CD107a=cluster of differentiation 107a; RTX-240=RTX-4-1BBL-IL-15TP product candidate.
• RTX-240 promotes NK cell cytotoxicity by increasing their ability to kill the myeloid leukemia cell line K562
• The cytotoxicity of NK cells was accompanied by increased degranulation (CD107a+) and activation of NK cells (CD69+)
Figure 7: mRBC-240, an RTX-240 Mouse Surrogate, Expands Functional NK and CD8 T Cells in CT26 Colorectal Tumor Model
Balb/c mice were inoculated with the CT26 colorectal cancer cell line (1x105 cells) subcutaneously on Day 0. Once tumors reached about 80 mm3, mice were administered 1x109 mRBC-CTRL, mRBC-4-1BBL,
mRBC-IL-15TP or mRBC-240 cells intravenously. Similarly, 4-1BB agonistic antibody (3H3) and rIL-15 were used as positive controls and administered intraperitoneally. On Day 4 post dosing, blood was collected
from each mouse, and the NK cell count (A), the percentage of cytotoxic NK cells (granzyme B+) (B) and proliferative NK cells (Ki67+) (C), CD8+ T cell count (D), and the percentage of proliferative CD8+ T cells (E),
were evaluated by flow cytometry. One-way Anova compared to mRBC-CTRL *P≤0.05, **P≤0.01, ****P≤0.0001.
GzmB=granzyme B; mRBC-240=mRBC-4-1BB+IL-15TP (mouse surrogate of RTX-240 generated using click chemistry); mRBC-CTRL=mRBC control; mRBC=murine red blood cells; NK cells=natural killer
cells; rIL-15=recombinant IL-15.
.
• mRBC-240 expanded NK cells and CD8 T cells in the blood as early as Day 4 in a CT26 subcutaneous colorectal cancer tumor model
• NK cells from mRBC-240-treated animals are cytotoxic and proliferative
• CD8 T cells from the mRBC-240-treated animals are highly proliferative
INTRODUCTION
Agonist antibodies and recombinant cytokines have had limited success in the clinic due to three factors: severe toxicity leading to a narrow therapeutic index, the diminished activity of an agonistic antibody compared with the natural ligand, and the lack of multiple signals needed to effectively activate most cell types. To address these limitations, Rubius Therapeutics has developed RTX-240, an allogeneic cellular therapy using genetically engineered red blood cells to express 4-1BBL and IL-15/IL-15Ra fusion (IL-15TP) in their natural conformation on the cell surface. RTX-240 is designed to mimic human biology by broadly stimulating adaptive and innate immunity to generate an anti-tumor response and provide improved safety due to the restricted biodistribution of red blood cells to the vasculature. Here we demonstrate that RTX-240 is highly active in preclinical models.
Figure 1: The RED PLATFORM® is Designed to Generate Allogeneic, Off-the-Shelf Cellular Therapies
• 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 enucleated red blood cells that express hundreds of thousands of copies of biotherapeutic proteins on the cell surface
• Universal, scalable, and consistent manufacturing process
OBJECTIVES
• To assess the immunostimulatory and anti-tumor activity of RTX-240 in vitro and in vivo
RUBIUS THERAPEUTICS TERMINOLOGY
• RCT=experimental construct
• RTX=Red Cell Therapeutic™ product candidate
• mRBC=mouse surrogate experimental construct
Figure 2: RTX-240 Mediates Immunostimulatory Effects Through Simultaneous Presentation of the Costimulatory 4-1BBL and the Cytokine IL-15TP
• RTX-240 is an allogeneic cellular therapy product candidate that simultaneously presents hundreds of thousands of copies of the costimulatory 4-1BB ligand (4-1BBL) and the trans-presented cytokine interleukin-15 (IL-15TP) in their native forms to activate and expand T and natural killer (NK) cells
• 4-1BBL is a costimulatory molecule that can drive T and NK cell proliferation and activation and interferon γ (IFNγ) production
• IL-15 is a cytokine that bridges innate and adaptive immunity by promoting T and NK cell proliferation, and NK cell cytotoxicity. IL-15TP is a fusion of IL-15 and IL-15 receptor alpha
Figure 3: RTX-240 Promotes Superior Activation and Expansion of NK Cells and CD8 T Cells Compared to Agonistic 4-1BB Ab and rIL-15 In Vitro
(A) PBMCs were labeled with CellTrace™ Far Red (CTFR) dye and cultured with engineered red blood cells starting at 5:1 ratio either in the absence of anti-CD3 for 8 days (B, D-F) or in the presence
of anti-CD3 for 5 days (C, G, H). PBMCs from 3 donors were evaluated using flow cytometry and (B) NK cell counts, (C) CD8+ T cell counts, (D) the percentage of divided NK cells determined by CTFR
dilution, (E) percentages of TRAIL and (F) NKp44 on NK cells, (G) the percentage of divided CD8+ T cells determined by CTFR dilution, and (H) the percentage of cytotoxic CD8+ T cells determined by
the expression of granzyme B were evaluated using flow cytometry.
IL=interleukin; NK cells=natural killer cells; RCT-CTRL=untransduced control; rIL-15=recombinant IL-15; RTX-240=RTX-4-1BBL-IL-15TP product candidate; TRAIL=tumor necrosis factor-related
apoptosis-inducing ligand.
Treatment with RTX-240 dramatically enhances CD8 T cell and NK cell expansion, and these expanded cells are functional.
• RTX-240 induces a dramatic increase in total counts of NK and CD8 T cells compared with 4-1BB agonistic antibody and rIL-15 in vitro
• RTX-240 promotes increased proliferation (Ki67) and function (granzymeB) of NK cells and CD8 T cells similar to the effects observed with a 4-1BB agonist antibody plus rIL-15
• RTX-240 promoted increased activation of NK cells as shown by increased percentage of TRAIL and NKp44+ NK cells
Figure 4: NK Cell Functionality and Activation is Dependent on the Ratio of RCTs:NK Cells and Directly Correlated With the Percentage of 4-1BBL/IL-15TP Expressed on the Cell
Red blood cells engineered to express various levels of 4-1BBL and IL-15TP on the cell surface were designated as low RCT-4-1BBL-IL-15TP, medium RCT-4-1BBL-IL-15TP, and RTX-240, and represents
cells with increasing percentage of 4-1BBL and IL-15TP double positive RCTs, respectively. (A) Purified NK cells (5 x 105) from 5 different donors were incubated with low RCT-4-1BBL-IL-15TP, medium
RCT-4-1BBL-IL-15TP, and RTX-240 at a ratio of 3:1 (circle) or 12: 1 (square) RCT to NK cells for 8 days. (B) is a table highlighting the percentage of double positive 4-1BBL/IL-15TP RCTs in the specific
populations of low RCT-4-1BBL-IL-15TP, the medium RCT-4-1BBL-IL-15TP and RTX-240 that were used in this experiment. (C) represents flow cytometry plots of 4-1BBL and IL-15TP staining on each
RCT. After 8 days of co-culture, the NK cell count (D), the percentages of divided NK cells determined by CTFR dilutions (E), the percentages of NKp44 (F), and TRAIL (G) on NK cells were evaluated
using flow cytometry. Mean and standard deviation values are plotted across all 5 donors for each treatment. A two-way Anova with Bonferroni post test was used to compare all means within the
same treatment between doses. *P≤0.05, **P≤0.01 , ***P≤0.001, ****P≤0.0001.
IL= interleukin; NK cells=natural killer cells; RTX-240=RTX-4-1BBL-IL-15TP product candidate; TRAIL=tumor necrosis factor-related apoptosis-inducing ligand.
• RTX-240 directly enhanced NK cell proliferation and activation as shown by increased TRAIL and NKp44 expression on the surface of NK cells
• Activation and function of purified human NK cells are directly correlated with an increase in RCT to NK cell ratio
• Activation and function of purified human NK cells are directly correlated with an increase in the percentage of 4-1BBL/IL-15TP double positive RCTs
Figure 8: mRBC-240 is Effective in a B16F10 Tumor Model and is Associated With the Expansion of Terminally Differentiated NK Cells
C57BL/6 mice were inoculated with B16F10 melanoma cells (1x105 ) intravenously on Day 0. Mice were administered 1x109 mRBC-CTRL, mRBC-4-1BBL,
mRBC-IL-15TP, or mRBC-240 cells intravenously on Days 1, 4, and 8 after tumor inoculation. An agonistic 4-1BB antibody (3H3) and rIL-15 were used as positive
control and administered intraperitoneally on Days 1, 4, and 8 post tumor inoculation. (A) On Day 14, mice were euthanized and the number of lung metastases
were quantified by counting metastases in the lung, (B) depicts the spleen weights from the different mice harvested on Day 14. Lungs from the different mice
were harvested for flow cytometry on Day 14, (C) the percentage of terminally differentiated NK cells (defined as CD11b+CD27-KLRG1+) in the lung is reported, and
(D) a Spearman rank correlation depicts the relationship between the percentage of terminally differentiated NK cells and the number of lung metastases at Day
14. One-way Anova compared to mRBC-CTRL. **P≤0.01, ****P≤0.0001.
CD11b=cluster of differentiation 11b; CD27=cluster of differentiation 27; KLRG1=Killer cell lectin-like receptor subfamily G member 1; NK cells=natural killer cells;
mRBC-CTRL=mRBC control; mRBC-240=mRBC-4-1BB-IL-15TP (a mouse surrogate of RTX-240 generated using click chemistry); rIL-15=recombinant IL-15.
• mRBC-240 was effective in reducing the number of lung metastases in a B16F10 intravenous lung metastases model
• Treatment with mRBC-240 did not lead to an increase in spleen weight as opposed to 4-1BB agonistic antibodies and rIL-15
• mRBC-240 expanded terminally differentiated NK cells, and their frequency is associated with efficacy in the B16F10 melanoma model
CONCLUSIONS
• RTX-240 increased CD8 T cell and NK cell expansion and activation in vitro when compared to 4-1BB agonistic antibody and rIL-15, and this was directly correlated with the percentage of 4-1BBL and IL-15TP expressed on the cell surface
• RTX-240 expanded CD56dim NK cells in vitro, a population with known cytotoxicity
• RTX-240 promoted NK cell killing of a myeloid leukemia cell line, K562, and this was accompanied by increased NK cell degranulation and activation
• A murine surrogate for RTX-240, mRBC-240, promoted significant expansion of CD8 T cells and NK cells in vivo as early as day 4 post-treatment in a murine model of CT26 colorectal cancer
• mRBC-240 demonstrated potent antitumor activity in a B16F10 melanoma model that was directly correlated with the expansion of terminally differentiated NK cells in the tumors
• RTX-240 is being evaluated in two Phase 1 clinical trials for the treatment of patients with relapsed/ refractory or locally advanced solid tumors and in relapsed/refractory acute myeloid leukemia
ACKNOWLEDGEMENTS
Poster design support was provided by Dennig Marketing Group, sponsored by Rubius Therapeutics.
DISCLOSURES
All authors: Employment with and equity ownership in Rubius Therapeutics.
CellTrace™ and LIVE/DEAD™ are trademarks of Thermo Fisher Scientific.
RESULTS
RCT-CTRL RCT-4-1BBL RCT-IL-15TP RTX-240 4-1BB agonist antibody rIL-15 rIL-15+4-1BB agonist antibody
0
20
40
60
80
100
%T
RA
IL+
of N
K C
ells
0
10
20
30
40
50
%N
Kp
44+
of N
K C
ells
0
20
40
60
80
100
120
%D
ivid
ed o
f CD
8 T
Cel
ls
0
20
40
60
80
100
%G
ZM
B+
of C
D8
Ce
lls
PBMCs
Dendritic Cell
B Cell
RTXs
+RTXs
T Cell
NK Cell
PBMC ASSAY B C
0
5,000
10,000
15,000
20,000
Num
ber
of N
K C
ells
NK Cell Count
%TRAIL+ of NK Cells CD8+ T Cell Proliferation % GranzymeB+ CD8+ T Cells%NKp44+ of NK Cells
0
20,000
40,000
60,000
80,000
Nu
mb
er o
f CD
8 T
Cel
ls
CD8+ T Cell CountA
E F
0
20
40
60
80
100
%D
ivid
ed o
f NK
Ce
lls
NK Cell ProliferationD
HG
NK Cell
T Cell
4-1BBL
4-1BBL
IL-15TP
IL-15TP
MIMICS HUMAN BIOLOGY: SIGNAL 2+3 RTX-240 (4-1BBL+IL-15TP)
4-1BBL
↑ NK proliferation/activation
↑ CD4 and CD8 proliferation/activation
IL-15 TP (IL-15/IL-15Rα fusion)
Proliferation/Survival
↓ TCR threshold
↓ Treg inhibition
NK CellCD8 T Cell
CD8 MEMORY
↑ Proliferation
↑ Cytotoxicity
NK CELL
RED PLATFORM®
ONE �HEALTHY�O- DONOR
EXPANSION & �DIFFERENTIATION
PROGENITOR �CELL COLLECTION
LENTIVIRAL VECTORENCODING OF A �
CO-STIMULATORYMOLECULE & CYTOKINE
ENUCLEATION & MATURATION
100-1000’s �OF DOSES
RED CELL THERAPEUTIC
Purified NK Cells
RTXs
+RTXs
NK Cells
% 4-1BBL/IL-15TP Double positive cells
IL-1
5Rα
PE
4-1BBL APC
Q113.0
Q22.44
Q35.96
Q478.6
0 103 104 105 106 107
RL1-H :: APC -a41BBL-H
0
103
104
105
106
107
BL2-
H ::
PE
aMs-
IL15
ra-H
Q136.7
Q222.9
Q310.7
Q429.7
0 103 104 105 106 107
RL1-H :: APC -a41BBL-H
0
103
104
105
106
107
BL2-
H ::
PE
aMs-
IL15
ra-H
Q132.3
Q253.0
Q34.76
Q49.99
0 103 104 105 106 107
RL1-H :: APC -a41BBL-H
0
103
104
105
106
107
BL2-
H ::
PE
aMs-
IL15
ra-H
0
500
1000
1500
2000****
*
Low Medium RTX-240
RCT-4-1BBL+IL-15TP
8-DAY NK CELL ASSAY B CA
0
20
40
60
80
100
% D
ivid
ed *
***
Low Medium RTX-240
RCT-4-1BBL+IL-15TP
% Divided NK Cells E
Cel
l Co
unt
NK Cell CountD
0
20
40
60
% T
RA
IL+
****
**
Low Medium RTX-240
RCT-4-1BBL+IL-15TP
% TRAIL+G
0
10
20
30
40
50
% N
Kp
44+
*
**
Low Medium RTX-240
RCT-4-1BBL+IL-15TP
% NKp44+F
Cells
Low RCT-4-1BBL-IL-15TP 2.44
22.9
53.9
Medium RCT-4-1BBL-IL-15TP
RTX-240
Low RCT-4-1BBL-IL-15TP
Medium RCT-4-1BBL-IL-15TP
RTX-240
Purified NK cells using negative selection
RTXs+
RTXs
NK Cells
CD
56
CD16RCT-CTRL RTX-240 rIL-15
0
2000
4000
6000
8000
Co
unt C
D56
dim
NK
Cel
ls
CD56dim NK Cells
**
CD56 dim98.2
CD56bright0.23
CD56 dim89.4
CD56bright7.44
CD56 dim69.1
CD56bright28.8
8-DAY NK CELL ASSAY B CA RCT-CTRL RTX-240 rIL-15
Purified NK cells using negative selection
RTXs+
RTXs
NK Cells
RTXs
+K562
NK Cells
NK Cell
K562
K562
NK Cell Degranulation
Control RTX-2400
10
20
30
40
50
% C
D10
7a+
**
Control RTX-2400
10
20
30
40
% C
D6
9+
% CD69 of NK Cells
**
Target Only Donor 1 Donor 2 Donor 3 Donor 4 Donor 50
20
40
60
80
% T
arg
et D
eath
% Dead K562 Targets
Control
RTX-240
CYTOTOXICITY ASSAY B
D
CA
mRBC-C
TRL
mRBC-4
-1BBL
mRBC-IL
-15T
P
mRBC-2
40
4-1B
B agonist antib
odyrIL
-15
mRBC-C
TRL
mRBC-4
-1BBL
mRBC-IL
-15T
P
mRBC-2
40
4-1B
B agonist antib
odyrIL
-15
mRBC-C
TRL
mRBC-4
-1BBL
mRBC-IL
-15T
P
mRBC-2
40
4-1B
B agonist antib
odyrIL
-15
mRBC-C
TRL
mRBC-4
-1BBL
mRBC-IL
-15T
P
mRBC-2
40
4-1B
B agonist antib
odyrIL
-15
mRBC-C
TRL
mRBC-4
-1BBL
mRBC-IL
-15T
P
mRBC-2
40
4-1B
B agonist antib
odyrIL
-15
0
10000
20000
30000
40000
50000
NK
Cel
l Co
unt
********
**
0
20
40
60
80
100
%G
ZM
B+
of N
K C
ells
********
****
0
20
40
60
80
100
%K
i67
of N
K C
ells
********
******
0
1000
2000
3000
CD
8+ T
Cel
l Co
unt
******
*
0
20
40
60
80
100
%K
i67
of C
D8+
T c
ells ****
********
NK Cell Count GranzymeB+ NK Cells NK Cell Proliferation CD8+ T Cell Count CD8+ T Cell Proliferation
Day 4 in CT26 Colorectal Tumor Model
B D ECA
mRBC-C
TRL
mRBC-4
-1BBL
mRBC-IL
15TP
mRBC-2
40
4-1B
B agonist antib
odyrIL
-15
mRBC-C
TRL
mRBC-4
-1BBL
mRBC-IL
15TP
mRBC-2
40
4-1B
B agonist antib
odyrIL
-15
mRBC-C
TRL
mRBC-4
-1BBL
mRBC-IL
15TP
mRBC-2
40
4-1B
B agonist antib
odyrIL
-15
0
50
100
150
200
# L
ung
Met
asta
ses
********
******
****
0.00
0.05
0.10
0.15
0.20
0.25
Sp
leen
Wei
ght
(g)
******
0
20
40
60
% N
K1.
1+ C
D11
b+
CD
27-
KLR
G1+
****
**
********
0
50
100
150
200
0
10
20
30
40
% N
K1.
1+ C
D11
b+
CD
27-
KLR
G1+
# Lung Metastases
p<0.0001
Day 14 in B16F10 i.v. Lung Metastases
Spleen WeightB D% Terminally Differentiated NK Cells
Correlation Between Efficacy and % Terminally
Differentiated NK Cells
CEfficacyA
