A CD45-targeted Antibody Drug Conjugate Enables ...... #EBMT20 Sharon L. Hyzy1, Rahul Palchaudhuri1,...
1
www.ebmt.org #EBMT20 Sharon L. Hyzy 1 , Rahul Palchaudhuri 1 , Jennifer L. Proctor 1 , Brad R. Pearse 1 , Ganapathy N. Sarma 1 , Geoff O. Gillard 1 , Asim Saha 2 , Tahirih L. Lamothe 1 , Melissa Brooks 1 , Katelyn Hammond 1 , Anjali Bhat 1 , Nicholas Clark 1 , Charlotte F. McDonagh 1 , Hans-Peter Kiem 3 , John E. Wagner 2 , Bruce R. Blazar 2 , Anthony E. Boitano 1 , Michael P. Cooke 1 (1) Magenta Therapeutics, Cambridge, MA, (2) Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN, (3) Fred Hutchinson Cancer Research Center, Seattle, WA Allogeneic hematopoietic stem cell transplant (allo-HSCT) is a potentially curative treatment for malignant and non-malignant blood disorders and has demonstrated impressive outcomes in autoimmune diseases. Current regimens for patient preparation, or conditioning, prior to allo-HSCT limit the use of this curative procedure due to regimen-related mortality and morbidities, including risks of organ toxicity, infertility, and secondary malignancies. This greatly limits the use of allo-HSCT in malignant and non-malignant conditions. To address these issues, we are developing novel antibody drug conjugates (ADCs) to provide the benefit of full- intensity conditioning to remove disease-causing cells while reducing the severity of treatment-related adverse events. To model these safer alternative conditioning strategies we have developed a novel ADC targeting mouse CD45, engineered to have rapid clearance, to provide a readily translatable approach that may enable allo-HSCT as a single agent. Previous work using an ADC approach to mouse CD45 has been shown to be sufficient to enable bone marrow transplant in syngeneic immune competent mice (Palchaudhuri et al. Nature Biotech 2016), and this approach in combination with CD4 and CD8 depleting antibodies has successfully enabled allo-HSCT in the haploidentical mouse model. The aim of this study was to model our engineered ADC to determine if it can be used to enable full allo-HSCT in mice. INTRODUCTION RESULTS • A single dose of the tool CD45-ADC is fully myeloablative and enables complete chimerism in a full mismatch allo-HSCT model. • See OS5-4 for additional work with this mouse tool CD45-ADC to enable immune reset with HSCT in mouse models of autoimmune disease. • This targeted, readily translatable approach for safer conditioning could improve the risk-benefit profile for allogenic and haploidentical HSCT and may extend the curative potential of this modality. CONCLUSION A CD45-targeted Antibody Drug Conjugate Enables Allogeneic Hematopoietic Stem Cell Transplantation as a Single Agent in Mice METHODS The CD45-ADC was evaluated in unmanipulated C57BL/6 mice to determine a myeloablative dose and to establish pharmacokinetics. The optimal dose of CD45-ADC was evaluated for the ability to condition for transplant in a congenic autologous mouse transplant model. Then, CD45-ADC was evaluated in an allogenic minor mismatch HSCT model in which conditioned DBA/2 mice were transplanted with 2 x 10 7 whole bone marrow cells harvested from pooled Balb/c CD45.1+ donors. Finally, we evaluated whether a single dose of the tool CD45-ADC was sufficient to enable chimerism in a full mismatch allo-HSCT model in which conditioned C57BL/6 mice (H2-b) were transplanted with 4 x 10 7 whole bone marrow cells from pooled Balb/c CD45.1+ (H-2d) donors. 9 Gy TBI served as the conventional conditioning positive control in all experiments. Peripheral blood chimerism assessed over 16 weeks. Anti-Mouse CD45-ADC Payload: Kills Rapidly Cycling Cells Target: Mouse CD45 Immune and HSC depletion Payload Engineering: Fc engineered for rapid clearance in mice to enable transplant ADCs Magenta’s platform enables the generation of targeted ADCs with customizable profiles. We developed a tool anti-mouse CD45 ADC engineered to have a short half-life (t 1/2 = 1.7hr, Figure 1 B) to enable HSCT. Animal studies C57BL/6, DBA/2, B6.SJL (B6 CD45.1+), and CbyJ.SJL (Balb/c CD45.1+) mice were used in these studies. Figure 1: CD45-ADC effectively depletes murine HSCs and lymphocytes. CD45-ADC was dosed on day 0. Bone marrow was collected on day 2 and HSC depletion assessed by flow cytometry. (A) Phenotypic long-term HSC (LT-HSC) depletion 2 days after single dose of CD45-ADC administration. (B) % LT-HSC depletion. (C) CD45-ADC (3 mg/kg) half-life in C57Bl/6 mice is 1.7 hours. (D) Peripheral lymphocytes reach nadir by day 9 post administration of CD45-ADC (3 mg/kg), indicating effective depletion by CD45-ADC. *p <0.05 when comparing CD45-ADC treated mice versus untreated mice. A D C Bone Marrow HSC Content Peripheral Lymphocytes PBS CD45-ADC 3 mg/kg LT-HSC Depletion MPP LT-HSC 0.000 0.005 0.010 0.015 0.020 LT-HSC (% of Cells) Untreated 3 0.3 1 3 CD45-ADC Isotype-ADC mg/kg * * 0 5 10 15 Lymphocytes (10 3 cells/µ L) untreated 3 0.3 1 3 CD45-ADC Isotype -ADC Day 0 Day 3 Day 7 Day 9 Day 14 Day 21 mg/kg B 0 10 20 30 40 1 10 100 1000 10000 100000 Hours Post Administration Plasma Antibody Concentration (ng/mL) LLOQ Dose AUC inf / Dose Cmax / Dose Half-life Cl Vss mg/kg (hour*kg*µg/mL/mg) (kg*ug/mL/mg) (hours) (mL/hour/kg) (mL/kg) 3 35.6 13.7 1.73 28.1 69 c-kit c-kit CD48 CD48 Sca-1 Sca-1 CD150 CD150 Murine HSC depletion by CD45-ADC Murine Congenic Transplant 0 25 50 75 100 %CD45.1 of CD11b+ 9 Gy TBI 3 Isotype -ADC mg/kg 0.3 1 3 CD45-ADC 0 25 50 75 100 %CD45.1 of B220+ 9 Gy TBI 3 Isotype -ADC mg/kg 0.3 1 3 CD45-ADC 0 25 50 75 100 %CD45.1 of CD3+ 9 Gy TBI 3 Isotype -ADC mg/kg 0.3 1 3 CD45-ADC 0 25 50 75 100 % Donor Chimerism (CD45.1+) 9 Gy TBI 3 Isotype -ADC mg/kg 0.3 1 3 CD45-ADC Week 4 Week 8 Week 12 Week 16 Week 0 Figure 2: Single dose of CD45-ADC enables congenic transplant in murine model. C57Bl/6 mice were conditioned with 9 Gy TBI, Isotype- ADC, or CD45-ADC and transplanted with whole bone marrow from B6.SJL (B6 CD45.1+) mice. (A) Peripheral donor chimerism is >85% in mice conditioned with CD45- ADC (3 mg/kg) through 16 weeks post transplant, comparable to mice conditioned with 9 Gy TBI. (B-D) Peripheral donor engraftment is multilineage. Donor Chimerism Myeloid Chimerism B Cell Chimerism T Cell Chimerism C A D B Donor Chimerism Myeloid Chimerism B Cell Chimerism T Cell Chimerism C A D B Murine Minor Mismatch Transplant Murine Allogeneic Transplant 0 25 50 75 100 % CD45.1 of CD11b+ CD45-ADC Isotype-ADC 9 Gy TBI 0 25 50 75 100 % CD45.1 of B220+ CD45-ADC Isotype-ADC 9 Gy TBI 0 25 50 75 100 % CD45.1 of CD3+ 9 Gy TBI CD45-ADC Isotype-ADC 0 25 50 75 100 % Donor Chimerism (CD45.1) 9 Gy TBI CD45-ADC Isotype-ADC Figure 3: A single dose of CD45-ADC is sufficient to enable minor mismatch allogenic transplant of Balb/c CD45.1 donor cells into DBA/2 recipients. (A-D) C57BL/6 mice were conditioned with 3 mg/kg Isotype-ADC or CD45-ADC. CD45-ADC enables ≥ 95% donor chimerism (A) and peripheral donor engraftment through 16 weeks is multilineage (B-D). Figure 4: A single dose of 5 mg/kg CD45-ADC is sufficient to enable allogenic transplant of Balb/c CD45.1 donor cells into C57BL/6 recipients. (A-D) C57BL/6 mice were conditioned with 5 mg/kg Isotype-ADC or CD45-ADC. CD45-ADC enables ≥ 95% donor chimerism (A) and peripheral donor engraftment at 8 weeks is multilineage (B-D). 0 25 50 75 100 % Donor Chimerism (CD45.1+) 9 Gy TBI CD45-ADC Isotype-ADC 0 25 50 75 100 % CD45.1 of CD11b+ 9 Gy TBI CD45-ADC Isotype-ADC 0 25 50 75 100 % CD45.1 of B220+ 9 Gy TBI CD45-ADC Isotype-ADC 0 25 50 75 100 % CD45.1 of CD3+ 9 Gy TBI CD45-ADC Isotype-ADC Week 0 Week 4 Week 8 Week 12 Week 20 Donor Chimerism Myeloid Chimerism B Cell Chimerism T Cell Chimerism C A D B
Transcript of A CD45-targeted Antibody Drug Conjugate Enables ...... #EBMT20 Sharon L. Hyzy1, Rahul Palchaudhuri1,...
www.ebmt.org #EBMT20
Sharon L. Hyzy1, Rahul Palchaudhuri1, Jennifer L. Proctor1, Brad R. Pearse1, Ganapathy N. Sarma1, Geoff O.
Gillard1, Asim Saha2, Tahirih L. Lamothe1, Melissa Brooks1, Katelyn Hammond1, Anjali Bhat1, Nicholas
Clark1, Charlotte F. McDonagh1, Hans-Peter Kiem3, John E. Wagner2, Bruce R. Blazar2, Anthony E. Boitano1,
Michael P. Cooke1
(1) Magenta Therapeutics, Cambridge, MA, (2) Blood and Marrow Transplantation, Department of Pediatrics,
University of Minnesota, Minneapolis, MN, (3) Fred Hutchinson Cancer Research Center, Seattle, WA
Allogeneic hematopoietic stem cell transplant (allo-HSCT) is a potentially
curative treatment for malignant and non-malignant blood disorders and
has demonstrated impressive outcomes in autoimmune diseases. Current
regimens for patient preparation, or conditioning, prior to allo-HSCT limit
the use of this curative procedure due to regimen-related mortality and
morbidities, including risks of organ toxicity, infertility, and secondary
malignancies. This greatly limits the use of allo-HSCT in malignant and
non-malignant conditions. To address these issues, we are developing
novel antibody drug conjugates (ADCs) to provide the benefit of full-
intensity conditioning to remove disease-causing cells while reducing the
severity of treatment-related adverse events. To model these safer
alternative conditioning strategies we have developed a novel ADC
targeting mouse CD45, engineered to have rapid clearance, to provide a
readily translatable approach that may enable allo-HSCT as a single
agent.
Previous work using an ADC approach to mouse CD45 has been shown
to be sufficient to enable bone marrow transplant in syngeneic immune
competent mice (Palchaudhuri et al. Nature Biotech 2016), and this
approach in combination with CD4 and CD8 depleting antibodies has
successfully enabled allo-HSCT in the haploidentical mouse model. The
aim of this study was to model our engineered ADC to determine if it can
be used to enable full allo-HSCT in mice.
INTRODUCTION
RESULTS
• A single dose of the tool CD45-ADC is fully myeloablative and enables
complete chimerism in a full mismatch allo-HSCT model.
• See OS5-4 for additional work with this mouse tool CD45-ADC to enable
immune reset with HSCT in mouse models of autoimmune disease.
• This targeted, readily translatable approach for safer conditioning could
improve the risk-benefit profile for allogenic and haploidentical HSCT and
may extend the curative potential of this modality.
CONCLUSION
A CD45-targeted Antibody Drug Conjugate
Enables Allogeneic Hematopoietic Stem Cell
Transplantation as a Single Agent in Mice
METHODS
The CD45-ADC was evaluated in unmanipulated C57BL/6 mice to determine a
myeloablative dose and to establish pharmacokinetics. The optimal dose of CD45-ADC was
evaluated for the ability to condition for transplant in a congenic autologous mouse
transplant model. Then, CD45-ADC was evaluated in an allogenic minor mismatch HSCT
model in which conditioned DBA/2 mice were transplanted with 2 x 107 whole bone marrow
cells harvested from pooled Balb/c CD45.1+ donors. Finally, we evaluated whether a single
dose of the tool CD45-ADC was sufficient to enable chimerism in a full mismatch allo-HSCT
model in which conditioned C57BL/6 mice (H2-b) were transplanted with 4 x 107 whole bone
marrow cells from pooled Balb/c CD45.1+ (H-2d) donors. 9 Gy TBI served as the
conventional conditioning positive control in all experiments. Peripheral blood chimerism
assessed over 16 weeks.
Anti-Mouse CD45-ADC
Payload:
Kills Rapidly Cycling Cells
Target: Mouse CD45
Immune and HSC depletion
Payload
Engineering:
Fc engineered for rapid clearance in mice to
enable transplant
ADCs
Magenta’s platform enables the generation of
targeted ADCs with customizable profiles. We
developed a tool anti-mouse CD45 ADC
engineered to have a short half-life (t1/2 =
1.7hr, Figure 1 B) to enable HSCT.
Animal studies
C57BL/6, DBA/2, B6.SJL (B6 CD45.1+), and
CbyJ.SJL (Balb/c CD45.1+) mice were used in
these studies.
Figure 1: CD45-ADC effectively depletes murine HSCs and lymphocytes. CD45-ADC was dosed
on day 0. Bone marrow was collected on day 2 and HSC depletion assessed by flow cytometry. (A)
Phenotypic long-term HSC (LT-HSC) depletion 2 days after single dose of CD45-ADC administration.
(B) % LT-HSC depletion. (C) CD45-ADC (3 mg/kg) half-life in C57Bl/6 mice is 1.7 hours. (D) Peripheral
lymphocytes reach nadir by day 9 post administration of CD45-ADC (3 mg/kg), indicating effective
depletion by CD45-ADC. *p <0.05 when comparing CD45-ADC treated mice versus untreated mice.
A
DC
Bone Marrow HSC Content
Peripheral Lymphocytes
PB
SC
D4
5-A
DC
3 m
g/k
g
LT-HSC
Depletion
MPP
LT-HSC
0.000
0.005
0.010
0.015
0.020
LT
-HS
C (
% o
f C
ells
)
Untreated 3 0.3 1 3
CD45-ADCIsotype-ADC
mg/kg
*
*
0
5
10
15
Lym
ph
ocyte
s (
10
3 c
ells/µ
L)
untreated 3 0.3 1 3
CD45-ADCIsotype-ADC
Day 0
Day 3
Day 7
Day 9
Day 14
Day 21
mg/kg
B
0 10 20 30 401
10
100
1000
10000
100000
Hours Post Administration
Pla
sm
a A
nti
bo
dy
Co
nc
en
tra
tio
n
(ng
/mL
)
LLOQ
Dose AUCinf / Dose Cmax / Dose Half-life Cl Vss
mg/kg (hour*kg*µg/mL/mg) (kg*ug/mL/mg) (hours) (mL/hour/kg) (mL/kg)
3 35.6 13.7 1.73 28.1 69
c-k
itc-k
it
CD
48
CD
48
Sca-1
Sca-1
CD150
CD150
Murine HSC depletion by CD45-ADC
Murine Congenic Transplant
0
25
50
75
100
%C
D45.1
of
CD
11b
+
9 GyTBI
3
Isotype-ADC
mg/kg0.3 1 3
CD45-ADC
0
25
50
75
100
%C
D45.1
of
B220+
9 GyTBI
3
Isotype-ADC
mg/kg0.3 1 3
CD45-ADC
0
25
50
75
100
%C
D45.1
of
CD
3+
9 GyTBI
3
Isotype-ADC
mg/kg0.3 1 3
CD45-ADC
0
25
50
75
100
% D
on
or
Ch
imeri
sm
(C
D45.1
+)
9 GyTBI
3
Isotype-ADC
mg/kg0.3 1 3
CD45-ADC
Week 4 Week 8 Week 12 Week 16Week 0
Figure 2: Single dose
of CD45-ADC enables
congenic transplant in
murine model. C57Bl/6
mice were conditioned
with 9 Gy TBI, Isotype-
ADC, or CD45-ADC and
transplanted with whole
bone marrow from
B6.SJL (B6 CD45.1+)
mice. (A) Peripheral
donor chimerism is
>85% in mice
conditioned with CD45-
ADC (3 mg/kg) through
16 weeks post
transplant, comparable
to mice conditioned with
9 Gy TBI. (B-D)
Peripheral donor
engraftment is
multilineage.
Donor Chimerism Myeloid Chimerism
B Cell Chimerism T Cell ChimerismC
A
D
B
Donor Chimerism Myeloid Chimerism
B Cell Chimerism T Cell ChimerismC
A
D
B
Murine Minor Mismatch Transplant
Murine Allogeneic Transplant
0
25
50
75
100%
CD
45.1
of
CD
11b
+
CD45-ADC Isotype-ADC9 Gy TBI
0
25
50
75
100
% C
D45.1
of
B220+
CD45-ADC Isotype-ADC9 Gy TBI
0
25
50
75
100
% C
D45.1
of
CD
3+
9 Gy TBI CD45-ADC Isotype-ADC
0
25
50
75
100
% D
on
or
Ch
imeri
sm
(C
D45.1
)
9 Gy TBI CD45-ADC Isotype-ADC
Figure 3: A single dose of
CD45-ADC is sufficient to
enable minor mismatch
allogenic transplant of Balb/c
CD45.1 donor cells into DBA/2
recipients. (A-D) C57BL/6 mice
were conditioned with 3 mg/kg
Isotype-ADC or CD45-ADC.
CD45-ADC enables ≥ 95%
donor chimerism (A) and
peripheral donor engraftment
through 16 weeks is
multilineage (B-D).
Figure 4: A single dose of 5
mg/kg CD45-ADC is sufficient
to enable allogenic transplant
of Balb/c CD45.1 donor cells
into C57BL/6 recipients. (A-D)
C57BL/6 mice were conditioned
with 5 mg/kg Isotype-ADC or
CD45-ADC. CD45-ADC enables
≥ 95% donor chimerism (A) and
peripheral donor engraftment at
8 weeks is multilineage (B-D).
0
25
50
75
100
% D
on
or
Ch
imeri
sm
(C
D45.1
+)
9 Gy TBI CD45-ADC Isotype-ADC0
25
50
75
100
% C
D45.1
of
CD
11b
+
9 Gy TBI CD45-ADC Isotype-ADC
0
25
50
75
100
% C
D45.1
of
B220+
9 Gy TBI CD45-ADC Isotype-ADC0
25
50
75
100
% C
D45.1
of
CD
3+
9 Gy TBI CD45-ADC Isotype-ADC
Week 0
Week 4
Week 8
Week 12
Week 20
Donor Chimerism Myeloid Chimerism
B Cell Chimerism T Cell ChimerismC
A
D
B
