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I L 8 s e c r e t i o n f o l l o w i n g D C D 4 7 b l o ... - a novel SIRPα-4-1BBL dual signaling...
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DSP107 - a novel SIRPα-4-1BBL dual signaling protein (DSP) for cancer immunotherapy Yosi Meir Gozlan 1 , Susan Hilgendorf 2 , Alexandra Aronin 1 , Yehudith Jitka Sagiv 1 , Liat Ben-Gigi-Tamir 1 , Shira Amsili 1 , Ami Tamir 1 , Iris Pecker 1 , Shirley Greenwald 1 , Ayelet Chajut 1 , Adam Foley-Comer 1 , Yaron Pereg 1 , Amnon Peled 3 , Michal Dranitzki Elhalel 4 & Edwin Bremer 2 1 KAHR-Medical, Jerusalem, Israel; 2 University Medical Center, Dept. of Hematology, Groningen, Netherlands; 3 Goldyne Savad Institute of Gene Therapy, & 4 Nephrology and Hypertenstion Department, Hadassah Hebrew University Medical Center, Jerusalem, Israel Background Results Results Summary and conclusions Dual Signaling Proteins (DSPs) DSPs are a fusion of extracellular domains of Type-I and Type-II membrane proteins. All TNF superfamily ligands are Type-II proteins, allowing them to be combined with a myriad of targeting and effector Type-I proteins. A DSP Targeting/Effector Trimerized DSP = Increased Activity Target Cell Type-I membrane protein Extracellular N-terminus Type-II membrane protein Extracellular C-terminus TNF-SF Ligand Targeting / Functional Domain Fusion Protein Engineering DSPs are created using ubiquitous genetic engineering and molecular biology tools. Potent therapeutic proteins can be created by prioritizing pairs that provide a synergistic effect, combining accurate targeting with high efficacy. Targeted dual therapeutic effect enhances efficacy and limits toxicity. DSPs act as both targeting and effector molecules. The DSP platform enables production of fusion proteins as trimers, a structure that is essential for TNF receptor family activation. DSP 107 (SIRPα - 4 - 1 BBL) B Type-I protein arm TNF-SF ligand (type-II) arm T cell/NK 4-1BB CD47 Cancer cell 41BB L SIRPα DSP 107 Scientific “Eat and Hit” Concept C DSP107 targets CD47 overexpressing tumors, simultaneously blocking macrophage inhibitory signals and delivering an immune costimulatory signal CD47 is overexpressed on many cancer cells, and binds SIRPα on immune phagocytic cells to produce a “don’t eat me” signal 41BB CD47 / SIRPα interaction inhibits macrophage activity Phagocyte (inactive) T-cell (inactive) Tumor DSP107 binds CD47 on cancer cells, blocking the “don’t eat me” signal and 41BB on T cells, providing co-stimulatory signal DSP107 binds CD47 on cancer cells, blocking “don’t eat me” signal DSP107 binds 4-1BB on T cells stimulating their anti tumor activation Targeted immune activation leading to macrophage and T- cell mediated tumor destruction Immune cell activation Phagocytosis & Apoptosis T-cell (active) T-cell (active) T-cell (active ) Macrophages ingesting cancer cells present tumor antigens and amplify response DSP107 administration DSP Composition • CD47 is overexpressed on cancer cells and binds SIRPα on phagocytes to produce a “don’t eat me” signal, allowing tumors to evade the immune system. • SIRPα arm of DSP107 binds to CD47 on tumor cells, blocks CD47, and removes “don’t eat me” signal allowing phagocytes to ingest tumor cells. • 4-1BBL binding to 4-1BB on T cells and NK cells, stimulates their expansion, cytokine production, and the development of cytolytic effector functions Targeted anti-tumor immune activation DSP107 targets CD47 expressing tumor cells by blocking the corresponding immune checkpoint inhibitory signals while simultaneously activating the immune cells via the 4-1BB:4- 1BBL pathway DSP107 produces targeted immune activation leading to macrophage and T-cell mediated tumor destruction DSP 107 Structural Analysis D Full atomic 3D model Schematic 3D model Schematic 3D model • SIRPα in grey ribbons • CD47 (SIRPα ligand) in green ribbons • 4-1BBL in Blue ribbons and 2 additional copies (trimer) in light blue DSP 107 Successfully Produced in Trimeric Form • DSP107 (SIRPα-4-1BBL) was produced in ExpiCHO system and purified on columns • Protein was analyzed in reducing, non-reducing and de-glycosylated conditions by SDS-PAGE and Western blot, using Coomassie blue staining, anti SIRPα and anti 4-1BBL antibodies DSP107 produced as a trimer at expected size (60 kDa for the monomer) and is glycosylated. E SIRPα - N terminus detection 4-1BBL - C terminus detection DSP107 was produced at expected size (60 KDa) and is glycosylated as expected SEC-MALS based analysis indicate that DSP107 was produced in the form of trimers DSP 107 Binds Its Targets CD 47 and 4 - 1 BB at high affinity and blocks SIRP α - CD 47 interaction F • DSP107 interaction with its human and cynomolgus counterpart ligands determined by SPR (Biacore). DSP107 binds with high affinities to both CD47 and 4-1BB from both species. DSP107 protein efficiently binds CD47 and 4-1BB and blocks SIRPα-CD47 interaction DSP107 blocks the interaction of SIRPα-CD47 with a low EC50 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0 5 10 15 20 25 CD47-SIRPa relative interaction (OD) DSP107 ug/ml EC50 = 2.1 nM Interaction with human CD47 and 4-1BB Traget receptor ka (1/(M∙sec)) kd (1/sec) KD (M) h-CD47 5.29E+05 7.88E-04 1.49E-09 c-CD47 6.13E+05 8.71E-04 1.42E-09 h-4-1BB 4.84E+05 1.47E-04 3.05E-10 c-4-1BB 7.99E+05 1.39E-04 1.74E-10 • ELISA plates were coated with recombinant hCD47. • DSP107 was added at different concentrations. • Biotinylated SIRPα was added. • SIRPα binding to CD47 quantitated using streptavidin HRP + TMB substrate DSP 107 Augments Phagocytic Uptake of Cancer Cells G • A- PBMCs of healthy donors mixed with tumor cells, pre-stained with vibrant-DiD, in the presence or absence of DSP107. Uptake of tumor cells by granulocytes evaluated using flow cytometry. • B- Co-cultured with DSP107 combined with Rituximab (αCD20) or Cetuximab (αEGFR) • C + D- Primary leukemic cells isolated from peripheral blood of AML patients. co- cultured with granulocytes from healthy donors in the presence of increasing concentrations of DSP107. DSP107 efficiently augment phagocytosis of different cancer cell lines and primary AML cells, and further augment phagocytosis in combination with therapeutic Abs DSP107 augments phagocytic uptake of lymphomas, carcinomas and leukemia cells A DSP107 augments phagocytic uptake of tumor cells when combined with Rituximab and Cetuximab B DSP107 augments phagocytic uptake of primary AML cells of five different patients D C DSP 107 Activates 4 - 1 BB Signaling and Enhances T cell Activation H DSP107 effectively activates 4-1BB signalling and augments T cell activation • A- ELISA plates were coated with CD47 (PB: plate bound) and then incubated with DSP107, soluble SIRPα, soluble 4-1BBL or both. Following incubation, HT1080 cells overexpressing 4- 1BB were added for 24 hours. These cells secrete IL8 upon binding and activation of the 41BB signaling pathway. Secreted IL8 is determined by ELISA. • B- Following CD47 coating, the plates are blocked using an anti CD47 antibody. Potency Assay 0 500 1000 1500 2000 2500 IL8 [pg/ml] CD47 PB+DSP107 w/o CD47 PB+DSP107 CD47 PB+SIRP CD47 PB+SIRP CD47 PB+4-1BBL DSP107 conc. [nM] 1.1 2.2 4.4 8.8 17 35 71 0 Potency Assay with Blocking Ab 0 1 .1 2 .2 4 .4 8 .8 17 35 71 0 500 1000 1500 2000 2500 IL8 [pg/ml] Blocking effect DSP107 conc. [nM] CD47 PB + DSP107 + αCD47 Ab. A B DSP107 augments human T cells activation as evident by increase in IFNγ secretion and CD25 expression CD25 expression % • C- Human PBMCs isolated from healthy donor peripheral blood and cultured for 40 hours with addition of different concentrations of DSP107 protein in the presence of anti-CD3 or anti-CD3 plus IL2; IFN-γ concentration in the cell supernatant evaluated by ELISA. • D + E- T cells isolated from peripheral blood of healthy donor were activated for 48 hours with sub-optimal concentrations of CD3+IL2 or αCD3/αCD28 dynabeads, in the presence of different concentrations of DSP107. CD25 expression was determined by flow cytometry. E D C Anti CD3 + Control DSP107 - 3 µg/mL αCD3+IL2 αCD3+αCD28 dynabeads DSP107 (µg/mL) DSP 107 Increases T - cell Induced Cancer Killing I • SNU387 – Hepatocellular cancer cell line labeled with mCherry fluorescent protein (red). • T cells isolated from peripheral blood of healthy donor and co-cultured with the labeled cancer cells in Incucyte machine, with sub-optimal concentrations of αCD3/αCD28 dynabeads, with or without addition of DSP107, in the presence of Caspase 3/7 dependent cleavage-activated GFP substrate (green). • Green and red spectrum images were taken in overlap every 1.5 hours for the next 5 days. • Killing of cancer cells is determined by overlap of green fluorescence (apoptotic cells) with red fluorescence (cancer cells). Results were analyzed by the Incucyte machine software. • A- assay design • B- no cell killing is detected when T cells are not present • C- DSP107 augment T cell mediated killing of SNU387 HCC cells DSP107 enhances T cell mediated killing of cancer cells C B A No augmentation of cell killing is detected when T cells are not present DSP107 augment T cell mediated killing of SNU387 HCC cells • DSP107 was successfully produced in the form of trimers and both sides bind their cognate counterpart with high affinity. • DSP107 blocks the interaction of SIRPα with CD47 and induces phagocytosis of cancer cells as a single agent • DSP107 further augment phagocytosis of cancer cells, when combined with targeted therapies. • DSP107 activates TNF-R signaling and when added to primary human immune cells, augments activation of T-cells and enhances T cell mediated killing of tumor cells. • We demonstrated the functional activity of DSP107, a novel DSP fusion protein, providing checkpoint blockade and TNF superfamily co-stimulation in a single molecule. • Dual targeting, by the two functional sides of DSP107, offers multiple functionalities that act simultaneously and may result in a synergistic effect • The DSP platform can be designed for selective tumor site or microenvironment targeting and is adaptable to most checkpoint targets Correspondence: [email protected] + 4-1BBL DSP107 concentration (µg/ml) + αCD3 + IL2 + αCD3/αCD28 dynabeads
Transcript of I L 8 s e c r e t i o n f o l l o w i n g D C D 4 7 b l o ... - a novel SIRPα-4-1BBL dual signaling...
DSP107 - a novel SIRPα-4-1BBL dual signaling protein (DSP) for cancer immunotherapyYosi Meir Gozlan1, Susan Hilgendorf2, Alexandra Aronin1, Yehudith Jitka Sagiv1, Liat Ben-Gigi-Tamir1, Shira Amsili1, Ami Tamir1, Iris Pecker1,
Shirley Greenwald1, Ayelet Chajut1, Adam Foley-Comer1, Yaron Pereg1, Amnon Peled3, Michal Dranitzki Elhalel4 & Edwin Bremer2
1KAHR-Medical, Jerusalem, Israel; 2University Medical Center, Dept. of Hematology, Groningen, Netherlands; 3Goldyne Savad Institute of Gene Therapy, & 4Nephrology and Hypertenstion Department, Hadassah Hebrew University Medical Center, Jerusalem, Israel
Background
Results
Results
Summary and conclusions
Dual Signaling Proteins (DSPs)
DSPs are a fusion of extracellular domains of Type-I and Type-IImembrane proteins. All TNF superfamily ligands are Type-II proteins,allowing them to be combined with a myriad of targeting and effectorType-I proteins.
A
DSP Targeting/Effector
Trimerized DSP = Increased Activity
Target Cell
Type-Imembrane protein
Extracellular N-terminus
Type-IImembrane protein
Extracellular C-terminus
TNF-SF LigandTargeting /
Functional Domain
Fusion Protein EngineeringDSPs are created using ubiquitous genetic engineering and molecular biology tools. Potent therapeutic proteins can be created by prioritizing pairs that provide a synergistic effect, combiningaccurate targeting with high efficacy.
Targeted dual therapeutic effect enhances efficacy and limits toxicity.DSPs act as both targeting and effector molecules.
The DSP platform enables production of fusion proteins as trimers, a structure that is essential for TNF receptor family activation.
DSP107 (SIRPα-4-1BBL)B
Type-I protein arm TNF-SF ligand (type-II) arm
T cell/NK
4-1BBCD47
Cancer cell
41BBL
SIRPα
DSP107 Scientific “Eat and Hit” ConceptC
DSP107 targets CD47 overexpressing tumors, simultaneously blocking macrophage inhibitory signals and delivering an immune costimulatory signal
CD47 is overexpressed on many cancer cells, and binds SIRPα on immune phagocytic cells to produce a “don’t eat
me” signal
41BB
CD47 / SIRPαinteraction inhibits
macrophage activity
Phagocyte(inactive)T-cell
(inactive)
Tumor
DSP107 binds CD47 on cancer cells, blocking the “don’t eat
me” signal and 41BB on T cells, providing co-stimulatory
signal
DSP107 binds CD47 on cancer cells, blocking “don’t eat me”
signal
DSP107 binds 4-1BB on T cells stimulating their anti tumor activation
Targeted immune activation leading to macrophage and T-
cell mediated tumor destruction
Immune cell activation
Phagocytosis & Apoptosis
T-cell(active)
T-cell(active)
T-cell(active
)
Macrophages ingesting cancer cells present tumor antigens and
amplify response
DSP107 administrationDSP Composition
• CD47 is overexpressed on cancer cells and binds SIRPα onphagocytes to produce a “don’t eat me” signal, allowingtumors to evade the immune system.
• SIRPα arm of DSP107 binds to CD47 on tumor cells, blocksCD47, and removes “don’t eat me” signal allowingphagocytes to ingest tumor cells.
• 4-1BBL binding to 4-1BB on T cells and NK cells, stimulatestheir expansion, cytokine production, and the developmentof cytolytic effector functions
Targeted anti-tumor immune activationDSP107 targets CD47 expressing tumor cells by blocking thecorresponding immune checkpoint inhibitory signals whilesimultaneously activating the immune cells via the 4-1BB:4-1BBL pathway
DSP107 produces targeted immune activation leading to macrophage and T-cell mediated tumor destruction
DSP107 Structural AnalysisD
Full atomic 3D model Schematic 3D model
Schematic 3D model• SIRPα in grey ribbons
• CD47 (SIRPα ligand) in green ribbons
• 4-1BBL in Blue ribbons and 2 additional copies (trimer) in
light blue
DSP107 Successfully Produced in Trimeric Form
• DSP107 (SIRPα-4-1BBL) was produced in ExpiCHO system and purified on columns
• Protein was analyzed in reducing, non-reducing and de-glycosylated conditions by SDS-PAGE and Westernblot, using Coomassie blue staining, anti SIRPα and anti 4-1BBL antibodies
DSP107 produced as a trimer at expected size (60 kDa for the monomer) and is glycosylated.
E
SIRPα - N terminus detection
4-1BBL - C terminus detection
DSP107 was produced at expected size (60 KDa) and is glycosylated as expected
SEC-MALS based analysis indicate that DSP107 was produced in the form of trimers
DSP107 Binds Its Targets CD47 and 4-1BB at high affinity and blocks SIRPα-CD47 interactionF
• DSP107 interaction with its human and cynomolgus counterpart ligands determined bySPR (Biacore). DSP107 binds with high affinities to both CD47 and 4-1BB from bothspecies.
DSP107 protein efficiently binds CD47 and 4-1BB and blocks SIRPα-CD47 interaction
DSP107 blocks the interaction of SIRPα-CD47
with a low EC50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
0 5 10 15 20 25
CD
47
-SIR
Pa r
elat
ive
inte
ract
ion
(O
D)
DSP107 ug/ml
EC50 = 2.1 nM
Interaction with human CD47 and 4-1BB
Traget receptor
ka (1/(M∙sec))
kd(1/sec)
KD (M)
h-CD47 5.29E+05 7.88E-04 1.49E-09
c-CD47 6.13E+05 8.71E-04 1.42E-09
h-4-1BB 4.84E+05 1.47E-04 3.05E-10
c-4-1BB 7.99E+05 1.39E-04 1.74E-10
• ELISA plates were coated with recombinant hCD47.
• DSP107 was added at different concentrations.
• Biotinylated SIRPα was added.
• SIRPα binding to CD47 quantitated using streptavidin HRP + TMB substrate
DSP107 Augments Phagocytic Uptake of Cancer CellsG
• A- PBMCs of healthy donors mixed with tumor cells, pre-stained with vibrant-DiD,in the presence or absence of DSP107. Uptake of tumor cells by granulocytesevaluated using flow cytometry.
• B- Co-cultured with DSP107 combined with Rituximab (αCD20) or Cetuximab(αEGFR)
• C + D- Primary leukemic cells isolated from peripheral blood of AML patients. co-cultured with granulocytes from healthy donors in the presence of increasingconcentrations of DSP107.
DSP107 efficiently augment phagocytosis of different cancer cell lines and primary AML cells, and further augment phagocytosis in combination with therapeutic Abs
DSP107 augments phagocytic uptake
of lymphomas, carcinomas and leukemia cells
A
DSP107 augments phagocytic uptake of
tumor cells when combined with Rituximab and
Cetuximab
B
DSP107 augments phagocytic uptake
of primary AML cells of five different
patients
DC
DSP107 Activates 4-1BB Signaling and Enhances T cell ActivationH
DSP107 effectively activates 4-1BB signalling and augments T cell activation
• A- ELISA plates were coated with CD47 (PB: plate bound) and then incubated with DSP107,soluble SIRPα, soluble 4-1BBL or both. Following incubation, HT1080 cells overexpressing 4-1BB were added for 24 hours. These cells secrete IL8 upon binding and activation of the41BB signaling pathway. Secreted IL8 is determined by ELISA.
• B- Following CD47 coating, the plates are blocked using an anti CD47 antibody.
Potency Assay
0
0.0
11
0.0
02
2
0.0
04
4
0.0
08
8
0.0
17
0.0
35
0.0
71
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
IL 8 s e c re tio n fo llo w in g in c re a s in g c o n c o f D S P -1 0 7 tre a tm e n t
(w /o D S P w a s h )
C o m p o u n d c o n c [ n M ]
IL8
[p
g/m
l]
C D 4 7 P B + D S P 1 0 7
w /o C D 4 7 P B + D S P 1 0 7
C D 4 7 P B + S IR P /4 -1 B B L
C D 4 7 P B + S IR P
C D 4 7 P B + 4 -1 B B L
0
0.0
11
0.0
02
2
0.0
04
4
0.0
08
8
0.0
17
0.0
35
0.0
71
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
IL 8 s e c re tio n fo llo w in g in c re a s in g c o n c o f D S P -1 0 7 tre a tm e n t
(w /o D S P w a s h )
C o m p o u n d c o n c [ n M ]
IL8
[p
g/m
l]
C D 4 7 P B + D S P 1 0 7
w /o C D 4 7 P B + D S P 1 0 7
C D 4 7 P B + S IR P /4 -1 B B L
C D 4 7 P B + S IR P
C D 4 7 P B + 4 -1 B B L
DSP107 conc. [nM]
1.1 2.2 4.4 8.8 17 35 710
Potency Assay with Blocking Ab
0
1.1
2.2
4.4
8.8 17
35
71
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
IL 8 s e c re tio n fo llo w in g C D 4 7 b lo c k e r A b
D S P - 1 0 7 c o n c [n M ]
IL8
[p
g/m
l]
C D 4 7 P B + D S P 1 0 7
C D 4 7 P B + C D 4 7 B 6 H 1 2 ( In v itro g e n )
Blo
ck
ing
eff
ec
t
DSP107 conc. [nM]
0
1.1
2.2
4.4
8.8
17
35
71
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
IL 8 s e c re tio n fo llo w in g C D 4 7 b lo c k e r A b
D S P - 1 0 7 c o n c [n M ]
IL8
[p
g/m
l]
C D 4 7 P B + D S P 1 0 7
C D 4 7 P B + C D 4 7 B 6 H 1 2 ( In v itro g e n )
Blo
ck
ing
e
ffe
ct
CD47 PB + DSP107 + αCD47 Ab.
A B
DSP107 augments human T cells activation as evident by increase in IFNγ secretion
and CD25 expression
CD
25
exp
ress
ion
%
• C- Human PBMCs isolated from healthy donor peripheral blood and cultured for 40 hourswith addition of different concentrations of DSP107 protein in the presence of anti-CD3 oranti-CD3 plus IL2; IFN-γ concentration in the cell supernatant evaluated by ELISA.
• D + E- T cells isolated from peripheral blood of healthy donor were activated for 48 hourswith sub-optimal concentrations of CD3+IL2 or αCD3/αCD28 dynabeads, in the presence ofdifferent concentrations of DSP107. CD25 expression was determined by flow cytometry.
ED
C
Anti CD3+
Control
DSP107 - 3 µg/mL
αCD3+IL2αCD3+αCD28
dynabeadsDSP107 (µg/mL)
DSP107 Increases T-cell Induced Cancer KillingI• SNU387 – Hepatocellular cancer cell line labeled with mCherry fluorescent protein (red).
• T cells isolated from peripheral blood of healthy donor and co-cultured with the labeledcancer cells in Incucyte machine, with sub-optimal concentrations of αCD3/αCD28dynabeads, with or without addition of DSP107, in the presence of Caspase 3/7 dependentcleavage-activated GFP substrate (green).
• Green and red spectrum images were taken in overlap every 1.5 hours for the next 5 days.
• Killing of cancer cells is determined by overlap of green fluorescence (apoptotic cells) withred fluorescence (cancer cells). Results were analyzed by the Incucyte machine software.
• A- assay design
• B- no cell killing is detected when T cells are not present
• C- DSP107 augment T cell mediated killing of SNU387 HCC cells
DSP107 enhances T cell mediated killing of cancer cells
CB
A
No augmentation of cell killing is
detected when T cells are not
present
DSP107 augment T cell mediated
killing of SNU387 HCC cells
• DSP107 was successfully produced in the form of trimers and bothsides bind their cognate counterpart with high affinity.
• DSP107 blocks the interaction of SIRPα with CD47 and inducesphagocytosis of cancer cells as a single agent
• DSP107 further augment phagocytosis of cancer cells, whencombined with targeted therapies.
• DSP107 activates TNF-R signaling and when added to primaryhuman immune cells, augments activation of T-cells and enhancesT cell mediated killing of tumor cells.
• We demonstrated the functional activity of DSP107, a novel DSPfusion protein, providing checkpoint blockade and TNFsuperfamily co-stimulation in a single molecule.
• Dual targeting, by the two functional sides of DSP107, offersmultiple functionalities that act simultaneously and may result ina synergistic effect
• The DSP platform can be designed for selective tumor site ormicroenvironment targeting and is adaptable to most checkpointtargets
Correspondence: [email protected]
+ 4-1BBL
DSP107 concentration (µg/ml)
+αCD3 + IL2 +αCD3/αCD28 dynabeads
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