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Chimeric antigen receptor macrophages (CAR-M) induce antitumor immunity and synergize with T cell checkpoint inhibitors in pre-clinical solid tumor modelsStefano Pierini, PhDCarisma Therapeutics
3675 Market St.PhiladelphiaCarismatx.com
Disclosure Information
I have the following financial relationships to disclose.Employee of:
Carisma Therapeutics Inc.
Solid tumor challenges for adoptive cell therapy
3. Target antigen heterogeneity• Inherent resistance • Antigen negative relapse
1. Trafficking & penetration• Extravasation• Homing• Penetration
2. Tumor microenvironment• Immunosuppression• Exhaustion • Metabolic challenges• Poor proliferation & persistence • Low antigen presentation• Low Th1 cytokines
Monocytes & macrophages home to tumors
Pollard JW, et al. Nat Rev Drug Discov. 2019. Klichinsky M. et al. Nature Biotechnology. 2020
Myeloid cells are abundant in solid tumors Adoptively transferred macrophages home to tumors
Tumor Lung
Spleen Liver
Biodistribution of IR-labeled human CAR-M(Gastric carcinoma xenograft model)
Ad5f35 induces CAR expression and promotes an M1 phenotype in murine MACs without affecting viability
UTD-M CAR-M-1000
0
5000
10000
15000
CD80 MFI
Nor
mal
ized
CD
80 M
FI
UTD-M CAR-M-1000
0
5000
10000
CD86 MFI
Nor
mal
ized
CD
86 M
FI
UTD-M CAR-M-100
0
200
400
MHC-II MFI
Nor
mal
ized
MH
C-II
MFI
UTD-M CAR-M-1000
-500
0
1000
CD206 MFI
Nor
mal
ized
CD
206
MFI
2 4 6 13 2 4 6 130
20
40
60
80
100
Live%
Live%
Days2 4 6 13 2 4 6 13
0
20
40
60
80
100
CAR-HER2+%
CAR-HER2+%
Days
Transduction
M0 (neutral) macrophage
M1 (pro-inflammatory) macrophage
2 4 6 13 2 4 6 130
10000
20000
30000
40000
50000
CAR-HER2 MFI
MFI
of A
PC in
Liv
e ce
lls
CAR-M bagUTD-M bag
M1 Markers M2 Marker
CARs redirect multiple macrophage effector functions
0 20 40 600
2
4
6
CT26-HER2+ Killing kinetics
Time (hrs)
Nor
mal
ized
gre
en fl
uore
scen
t int
ensi
ty Target only
UTD-M 10:1
CAR-M 3:1CAR-M 10:1
UTD-M 3:1
CAR-M 1:1
UTD-M 1:1
0 20 40 600
2
4
6
8
hr
Nor
mal
ized
gre
en fl
uore
scen
t int
ensi
ty
MC38-OVA+HER2+ + OTI T cellsKilling kinetics MC38HER2 Alone
MC38HER2 + OTI T cells
MC38HER2 + UTD-M + OTI T cellsMC38HER2 + CAR-MMC38HER2 + CAR-M + OTI T cells
UTD-M CAR-M0
20
40
60
80
MHC on B16-HER2+
% o
f MHC
-I an
d -II
MHC-IIMHC-I (H-2Kb)
CAR-M clears HER2+
target cellsCAR-M induce MHC expression
on tumor cells
CAR-M enhance tumor killing of transgenic TCR T cells
(OTI OVA model)
CAR-M shrink tumors and improve overall survival in immunocompetent mouse models
0 20 40 600
200
400
600
800
1000
Untreated
Days
Tum
or V
olum
e (m
m3)
0 20 40 600
200
400
600
800
1000
UTD-M
DaysTu
mor
Vol
ume
(mm
3)
0 20 40 600
200
400
600
800
1000
CAR-M
Days
Tum
or V
olum
e (m
m3)
CT26 (HER2)
HER2 IHC: HER2 3+
Tumor Subcutaneous, grown 15 days
Treatment Local administration
P=0.19
0 20 40 60 800
50
100
Days
Sur
viva
l pro
babi
lity
CTRL
UTD-M
CAR-M P=0.02
CAR-M lead to tumor control and improve OS against established CT26 (colon cancer) HER2+
n Alive @ Day 75
Median Survival
CTRL 9 0/9 (0%) 42 days
UTD-M 9 1/9 (11.1%) 46 days
CAR-M 8 5/8 (62.5%) Not reached
UTD: Untransduced; CAR: anti-HER2; OS: overall survival
CAR-M reprogram and activate the TME in immunocompetent pre-clinical models
CTRL
UTD-M
CAR-M0
2000
4000
6000
8000
CD3 counts
# of
live
CD
45+C
D3+
/ 1e
5 ce
lls
CTRL
UTD-M
CAR-M0
500
1000
1500
2000
CD8 count
# of
CD
3+C
D8+
/ 1e
5 ce
lls
CTRL
UTD-M
CAR-M0
1000
2000
3000
4000
CD4 count
# of
CD
3+C
D4+
/ 1e
5 ce
lls
CTRL
UTD-M
CAR-M0
500
1000
1500
2000
B cells count
# of
CD
45R
+ C
D3-
/ 1e
5 ce
lls
CTRL
UTD-M
CAR-M0
500
1000
1500
2000
NK cells count
# of
CD
49b+
CD
3- /
1e5
cells
CTRL
UTD-M
CAR-M0
500
1000
1500
DC count
# of
CD
11c+
MH
CII+
CTRL
UTD-M
CAR-M0
50
100
150
200
250
Tetramer counts
# of
gp7
0 T
cells
/ 1e
5 ce
lls
Increased infiltration of T cells Ex vivo TIL restimulation with gp70 Increased infiltration & activation of DCs
Increased infiltration of B cells and NK cellsCTRL
UTD-M
CAR-M0
200
400
600
800
1000
CD86+ DCs
# of
CD8
6+ D
Cs
CTRL UTD-M CAR-M0
2
4
6
8
10
TNFa%
of C
D8+
TNFa
lpha
+
No peptidegp70 peptide
CTRL UTD-M CAR-M0
10
20
30
40
IFN gamma
% o
f CD8
+ IF
Ngam
ma+
No peptidegp70 peptide
CAR-M drive T cell infiltration into solid tumors
Representative CD8 IHC CTRL UTD-M CAR-M
200um 200um 200um
Tumor Area Stroma Area0
500
1000
1500
2000
CD8 infiltration (Density)In
filtr
atio
n (p
ositi
ve c
ells
/mm
2 )ControlUTD-MCAR-M
Tumor Area Stroma Area0
500
1000
1500
CD3 infiltration (Density)
Infil
trat
ion
(pos
itive
cel
ls/m
m2 )
ControlUTD-MCAR-M
CAR-M therapy vaccinates mice against tumor recurrence and prevents antigen negative relapse
0 10 20 30 400
50
100
Days
Surv
ival
pro
babi
lity
(%) Naive BALB/c
CAR-M
CT26 Wt (HER2-) rechallenge:Kaplan Meir Survival Curve
CAR-M promote systemic anti-tumor response and enhance abscopal effect
Evaluation of the TME in the HER2- (abscopal) tumor
CAR-M + PD-1 blockade leads to improved tumor control and survival in immunocompetent animals
0 60 1200
50
100
Days
Sur
viva
l pro
babi
lity
(%) CTRL
CAR-M
CAR-M + aPD-1
aPD-1
-100
-50
00
250
500
4000
% c
hang
e in
tum
or v
olum
e re
lativ
e to
bas
elin
e
CTRL aPD-1 CAR-M CAR-M + aPD-1
0 10 20 30 40 50 600
400
800
1200
1600
Days
Tum
or V
olum
e (m
m3) Untreated
CAR-MCAR-M +aPD-1
aPD-1
P<0.01
anti-PD-1Days 14 23
Conclusions
§ CAR-M quickly eradicate tumor cells in a CAR- and HER2-specific way
§ CAR-M induce MHC expression on tumor cells and enhance immunorecognition of the target cells by T cells
§ Local administration of CAR-M modulates the TME, impacts primary and abscopal tumor growth and increases mice survival
§ CAR-M therapy vaccinates mice against tumor recurrence and prevents antigen negative relapse
§ The combination of CAR-M with anti-PD-1 blockade leads to synergistic tumor control and significantly increases overall survival
