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TCRL Generation
• MHC-peptide complex generation for selected peptide
• TCRL clones generation by standard hybridoma technology
• Lead clone selection by analysis of binding assays, Alanine Scan, SPR
• TCRL In Vitro/In Vivo validation in tumor models using CAR & Bi-Specific modalities
• TCRL - Target Moiety for immune cell product / other modalities
EpiTarget- Target Discovery
• Cancer and normal tissues peptide analysis by Mass Spectrometry to mine differential expression of cancer MHC peptides
• Target validation by cross correlating with normal tissue mRNA databases
• Target validation by targeted search of specific peptide by Mass Spectrometry and use of proprietary supportive analytical tools
• Peptide target selection for TCRL
Target Selection and Prioritization
Supportive tools
0
1000
2000
3000
4000
N_A
DIP
OS
E
N_B
LA
DD
ER
N_BRAIN-…
N_B
RO
NC
HU
S
N_D
UO
DE
NU
M
N_G
AL
LB
LA
DD
ER
N_IR
IS
N_LU
NG
N_MESOTHELIAL-…
N_P
AN
CR
EA
S
N_P
ITU
ITA
RY
N_S
ALIV
AR
Y-G
LA
ND
N_S
MA
LL-I
NT
ES
TIN
E
N_T
HY
RO
ID-G
LA
ND
N_U
RE
TE
R
N_B
LO
OD
-CE
LLS
N_O
VA
RY
N_S
PLE
EN
C_BLADDER_CARCI…
C_B
LA
DD
ER
_T
CC
C_B
LA
DD
ER
_T
CC
C_C
ER
VIX
_S
CC
C_C
ER
VIX
_S
CC
C_E
SO
PH
AG
US
_S
CC
C_LA
RY
NX
_S
CC
C_LUNG_ADENOCA…
C_LU
NG
_S
CC
C_LU
NG
_S
CC
C_OVARY_ADENOC…
C_OVARY_ADENOC…
C_O
VA
RY
_P
AP
ILL
AR
Y
C_OVARY_SEROUS…
C_SOFT-…
C_SOFT-…
C_T
ON
GU
E_S
CC
C_T
ON
GU
E_S
CC
C_UTERUS_ENDOM…
L_M
ELA
NO
MA
Normal Cancer
Protein libraries
Mutation libraries
Peptide libraries
Spectral libraries
RNA-seq repositories
Denovo sequencing
Differential presentation
analysis
mRNA expression
Databases
Synthetic peptides
Similarity analysis
Literature
Peptide presentation
Verified
Target
GTEx
Sum
mar
y
mRNA Expression in Various Cancers
Regression of Established Bladder Cancer Xenograft by MAGE-A4 TCRL-CD3 BiSpecific Compound
• Mel526 melanoma cells (5x106) inoculated into NOD/SCID mice with ex-vivo expanded T cells (E/T 3:1)
• Upon tumor establishment (70-100mm3) murine, humanized D11 or control Bispecific TCRLs administrated at days 5-14 at the indicated doses (15µg/dose x 10)
• Parental murine and humanized D11 Bispecific exhibit similar anti-tumor activity
Murine and Humanized D11 Tyr TCRL-CD3 Bispecific is Efficacious in Established Melanoma Tumor Model
Cancer &Normal tissues
Selection of cancer-specific candidate
peptides
Validation of candidate peptide targets
• SCaBER XCL bladder cancer cells (10x106) inoculated into NOD/SCID mice with ex-vivo expanded T cells (E/T 3:1)
• Upon tumor establishment (~200mm3) C106B9-CD3 Bispecific or control TCRLs administered at days 4-9 at the indicated doses (15µg/dose x 6)
Specific Killing of Multiple Tumor HLA-A2+/MAGE-A4(+) Cell Lines by C106B9 TCRL-CD3 Bispecific
TCRL Platform Technology: Accessing the Intracellular Proteome • Challenge: Paucity of disease-specific cell surface targets in solid tumors (80%
of proteins are intracellular)
• Solution: Targeting disease-specific intracellular proteins highly expands target pool
• TCRLs are specific to peptide-MHC complexes
TCR recognition of MHC-peptide
TCR
Multiple Application of TCRLs
– scFv for chimeric antigen receptors
– Bispecific T-cell engaging antibodies
– Antibody-drug conjugates
TCRL recognition of MHC-peptide
TCRL
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nM
ER
T101/ 1
nM
ER
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nM
ER
T101/ 3
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
M E R T 1 0 1
re
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e a
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nd
an
ce
(a
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3
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RT
102/ 7
nM
ER
T102/ 3
nM
ER
T102/ 2
nM
ER
T102/ 1
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
3 0 0 0 0 0
M E R T 1 0 2
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Normalskin
• Expressed in the majority of primary and metastatic melanoma (>70% by mRNA), including stage 3 and 4 disease
• Expression in normal tissues is limited to melanocytes, retina/choroid and inner ear
• Tyr peptide 369-377 Identified by Mass Spec in:o Melanoma patient specimens 6/8 (75%) and Melanoma cell lines 6/7 (86%) Tyr mRNA+ o Normal eye (retina, choroid and iris) and skin
Specific Killing of Melanoma HLA-A2+/Tyr+ Cell Lines but not Tyr- Cell Lines
Mela
no
ma
Ovary
Bla
dd
er
Gastr
o-e
so
ph
ag
us
H&
N
En
do
metr
ium
Lu
ng
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
3 0 0 0 0
6 0 0 0 0
1 2 0 0 0 0
1 3 5 0 0 0
1 5 0 0 0 0
EV
1
• Identified by Mass Spec in H&N SCC, Bladder TCC, Ovarian, Lung and Esophageal HLA-A2+ patient specimen
• In normal tissues, MAGE-A4 is expressed in placenta and testis.
T-cell receptor-like antibodies directed against intracellular tumor targets for immunotherapy of solid tumorsGalit Denkberg1; Ilan Beer1; Yael Elbaz Teboul1; Reut Segal-Erel1; Lyora A. Cohen1; Dotan Sela1; Daulet Satpayev2; Hui Shao2; Yoram Reiter1; Stewart E. Abbot2
1. Adicet Bio Israel Ltd., Haifa, Israel; 2. Adicet Bio Inc., Menlo Park, CA 94025 US
Critical to the success of immunotherapy of cancer is the ability toselectively target malignant cells. Peptides from intracellular proteins can bepresented on the surface of cells, via human leucocyte antigen (HLA)molecules. These peptide-HLA complexes are monitored and recognized by Tcell receptors (TCRs) expressed by T cells. The fine specificity of TCRs can bemimicked by monoclonal antibodies that exhibit similar peptide-specific, HLA-restricted recognition and are termed TCR-like antibodies (TCRLs).
Adicet has established a hybridoma-based platform to derive such TCR-likeantibodies and a robust TCRL validation process to isolate target-specificTCRLs. This process includes assessment of antibody affinity and specificity byscreening a large panel of irrelevant and similar peptides to ensure theselectivity of TCRLs and eliminate the potential for off-target cross reactivity.In addition, we have established a mass-spectrometry (MS) - based approach,the “EpiTarget platform”, to identify novel, disease-specific HLA / peptidecomplexes in patient tumor specimens. Here we present two highly specificTCRLs targeting two HLA-A*02-restricted peptides: Tyrosinase 369-377 (Tyr) andMAGE-A4230-239. These two target peptides were identified and validated byMS and are present in the majority of melanoma specimens (Tyr) and varioussolid tumors (MAGE-A4). Two highly specific TCRLs against HLA-A*02 / Tyr andHLA-A*02 / MAGE-A4 peptides were identified and converted into CD3-TCRLbispecific T-cell engager format. Both exhibit robust potency in vitro against apanel of target positive cell lines and in vivo in various xenograft models ofmelanoma and bladder cancer.
Introduction
Peptide/MHC epitope mapping demonstrates high degree of peptide-dependent interactions
Ala scanning analysis
No binding to multiple peptides with sequence similarity identified by whole proteome mining
No binding to multiple HLA-A2+ antigen negative cells A panel of different tumor cell lines and primary normal cells from 12
different essential tissue types
Potent specific in vitro & in vivo killing of HLA-A2+ Ag+ cells by TCRL-CD3 bi-specific MAb
Demonstrated on cell lines with low presentation levels of peptide-MHC complexes
High Affinity (nM range), High Specificity
Superior TCRLs
TCRL Selection - High Affinity and High Specificity
• Two HLA-A*02-restricted target peptides: Tyrosinase 369-377 (Tyr) and MAGE-A4230-239 were identified and validated by Mass Spectrometry to be presented inthe majority of melanoma specimens (Tyr) and various solid tumors (MAGE-A4)
• Two highly specific TCRLs against HLA-A*02 / Tyr and HLA-A*02 / MAGE-A4 peptides were identified and converted into CD3-TCRL bispecific T-cell engagerformat and exhibit robust potency in vitro against a panel of target positive cell lines and in vivo in xenograft models of melanoma and bladder cancer,respectively
• Identification and validation of additional novel intracellular targets by Epitarget analysis is expected to provide a rich pipeline for TCRL-based treatmentmodalities for cancer, such as bispecific T-cell engaging antibodies, ADC, and chimeric antigen receptor modified T cells directed against solid tumors
• The TCRL platform complements Adicet’s efforts to develop gamma delta T cell-based next generation immunotherapies
Tyrosinase is an Attractive Target for Melanoma MAGE-A4 as a Target for Multiple Solid Tumors
0102030405060
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
J82 (Bladder)
0102030405060
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
OVCAR (Ovary)
01020304050
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
HepG2 (Liver)
MAGE-A4 positive cells MAGE-A4 negative cells
0
10
20
30
40
50
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
SCaBER (Bladder)
01020304050
0 -12 -11 -10 -9 -8Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
A375 (Melanoma)
0
20
40
60
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
U2OS (Osteosarcoma)
Targ
et v
alid
atio
n
In-v
itro
effi
cacy
mRNA Expression in Melanoma and Normal Skin
Tyr expression in melanoma specimens (qPCR)
Targ
et v
alid
atio
n
In-v
ivo
effi
cacy
WM266.4 - positive control
Panc1 – negative control
Tested cells as indicated
01020304050
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (%
)
Log10 [D11 Bispecific] (M)
Mel 526
01020304050
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (%
)
Log10 [D11 Bispecific] (M)
SKMel5
01020304050
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (%
)
Log10 [D11 Bispecific] (M)
Malme-3M
0
20
40
60
0 -12 -11 -10 -9 -8Log10 [D11 Bispecific] (M)
A375 (Melanoma)
0
10
20
30
40
50
0 -12 -11 -10 -9 -8Log10 [D11 Bispecific] (M)
C33A (retinoblastoma)
0
10
20
30
40
50
0 -12 -11 -10 -9 -8
Log10 [D11 Bispecific] (M)
Saos-2 (Osteosarcoma)
0
10
20
30
40
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [hD11 Bispecific] (M)
ARPE-19 (retina)
0
10
20
30
40
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [hD11 Bispecific] (M)
Primary Melanocytes
0
10
20
30
40
50
60
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [hD11 Bispecific] (M)
ARPE-19 after diff.
Tyr positive cells Tyr negative cells
In-v
itro
effi
cacy
Primary cells
0.0
500.0
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1500.0
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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
MEA
N T
UM
OR
VO
LUM
E (M
M3
)
DAYS
mel526 only
mel526 + T cells + PBS
mel526 + T cells + hD11 TCRL (15µg/mouse)
mel526 + T cells + mD11 TCRL (15µg/mouse)
mel526 + T cells + control TCRL (15µg/mouse)
Primary cells
0
20
40
60
80
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
Renal epithelial cells
0
20
40
60
80
0 -12 -11 -10 -9 -8Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
Cardiac myocytes
0
20
40
60
80
0 -12 -11 -10 -9 -8
Cyt
oto
xici
ty (
%)
Log10 [C106B9 Bispecific] (M)
Urothelial cells
1938 – melanoma positive control
JEKO1 or Panc1 – negative control
Cell lines – as indicated
0.0
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6000.0
7000.0
0 5 10 15 20 25 30
Mea
n T
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Vo
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e (m
m3)
Days
ScaBER XCL only
ScaBER XCL + PBMCs + PBS
ScaBER XCL + PBMCs + C106B9 TCRL (15µg/mouse)
ScaBER XCL + PBMCs+ Control TCRL (15µg/mouse)
In-v
ivo
effi
cacy T cells
T cells
T cells
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