A Novel Targeted Approach to Achieve Immune System Reset ...€¦ · peripheral CD3+ cells from...
Transcript of A Novel Targeted Approach to Achieve Immune System Reset ...€¦ · peripheral CD3+ cells from...
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Figure 2: Immune Reset via anti-mouse CD45-ADC conditioning enables congenic engraftment, delays EAE onset,
reduces EAE incidence, and kills disease-mediating effector T cells in vivo
C57BL/6 (CD45.2+) mice were induced with MOG35-55 peptide in complete Freund’s adjuvant, followed by the administration
of pertussis toxin (PTX). Immunized Mice were conditioned for CD45.1+ congenic transplant (B6.SJL donor) on day 5 post-
immunization and transplanted 48 hours after conditioning (A). Terminal analysis of tissues (bone marrow, peripheral blood,
spleen, lymph nodes) were demonstrated that conditioning with anti mouse CD45-ADC (B) enabled full donor chimerism (C).
Global depletion of host hematopoietic cells with CD45-ADC included depletion of IL-17A-producing effector cells (D).
Disease onset was around 9-12 days post induction for vehicle- or CD45 antibody-treated animals, while treatment with
CD45-ADC substantially delayed disease onset and reduced disease incidence (E). A less substantial effect on donor
chimerism and disease-modification was observed in animals treated with isotype-ADC, a result consistent with known
platform toxicity of the tool ADC payload on rapidly cycling cells. In vitro, the human anti-CD45-ADC mediated efficient killing
of PBMC from MS patients (blue) and healthy donors (magenta) (F).
A Novel Targeted Approach to Achieve Immune System Reset: A Single Dose of Magenta CD45-Targeted Antibody Drug Conjugate Enables Autologous HSCT and Ameliorates Disease in Murine Models of Autoimmune Disease
Geoffrey O. Gillard, Jennifer L. Proctor, Melissa Brooks, Tahirih L. Lamothe, Sharon L. Hyzy, Sean McDonough, Rahul Palchaudhuri, Anjali Bhat, Ganapathy N.
Sarma, Prashant Bhattarai, Pranoti Sawant, Brad R. Pearse, Charlotte F. McDonagh, Anthony E. Boitano, Michael P. Cooke
Magenta Therapeutics, Cambridge, MA
BACKGROUND
CONCLUSIONS
IMMUNE RESET AFTER ANTI-MOUSE CD45-ADC
CONDITIONING AND BMT AMELIORATES DISEASE IN A MURINE
MODEL OF MULTIPLE SCLEROSIS
Immune reset was achieved via a single dose of CD45-ADC and BMT leading to substantial disease-
modifying effects in multiple murine models of autoimmune disease comparable to or better than approved
mechanisms.
Single dose administration of an anti-mouse tool CD45-ADC is well tolerated and enables congenic
transplant in mice.
The anti-mouse tool CD45-ADC has a dual mechanism of action in these models by (a) preferentially
removing the proliferating allogeneic T cells in vivo compared to resting T cells and (b) rendering the
remaining T cells incapable of responding to allogeneic cells and primed for killing.
An anti-human CD45-ADC kills T cells from MS patients in vitro. In vivo, a single dose of anti-human CD45-
ADC is well tolerated, results in elimination of disease-causing cells, and ameliorates pathology in a
scleroderma-like model of xenoGVHD in hNSG mice.Magenta Therapeutics
Animals: C57BL/6 (CD45.2+), B6.SJL (B6 CD45.1+), DBA/2, Balb/C (CD45.2+), and CbyJ.SJL (Balb/C CD45.1+)
mice were purchased from the Jackson Laboratories. All in vivo research was conducted in accordance with the
Guide for the Care and Use of Laboratory Animals published by the National Research Council of the National
Academies and under the approval of the Institutional Animal Care and Use Committee.
Congenic Transplant Model: Conditioned mice were transplanted with 2 x107 whole bone marrow cells harvested
from pooled B6.SJL(EAE) or BALB/c (PGIA) CD45.1+ congenic donors, and chimerism was assessed at the
indicated time points.
Resetting the immune system through autologous hematopoietic stem cell transplant (autoHSCT) is a highly effective
treatment in selected patients with autoimmune diseases. AutoHSCT can induce long-term remission (up to 15
years) with 80% progression free survival in patients with multiple sclerosis (Muraro 2017) that is superior to
standard of care agents (Burt 2019). Likewise, use of autoHSCT in scleroderma patients achieved superior
outcomes in two randomized studies (Tyndall 2014, Sullivan 2018). These impressive results are achieved by a
combination of the eradication of autoreactive immune effector cells and re-establishment of a self-tolerance, i.e.,
immune system reset. However, only a small fraction of eligible patients undergo autoHSCT due to toxicity
associated with current conditioning protocols. To address these issues, we are developing antibody drug conjugates
(ADCs) that selectively target CD45 to eradicate autoimmune cells and enable autoHSCT as a potential one-time
curative treatment for patients with autoimmune disease.
To model this approach in mice, we generated an anti-mouse CD45 ADC that was evaluated for the ability to
condition recipients in a murine congenic transplant model following a single myeloablative dose. This ADC was
further evaluated for its ability to eliminate pathogenic host-reactive cells and enable immune reset in recipients in
multiple murine models of autoimmune disease, including MOG-induced experimental autoimmune encephalitis
(EAE) and proteoglycan-induced arthritis (PGIA).
In EAE, conditioning with a myeloablative dose of 3 mg/kg of the CD45-ADC followed by congenic transplant prior to
disease onset led to full donor chimerism, significantly delayed disease onset, reduced overall disease incidence
(3/12; 25%) and reduced disease severity. In the EAE model, we also observed that the isotype ADC had a lesser
effect in this model (28 days to disease onset, 5/12 (40%) incidence), consistent with the platform toxicity of the
murine ADC payload on rapidly dividing cells in an immunization model. To further explore the specific targeting of
CD45 cells with the CD45-ADC vs. the isotype ADC, we explored the effect of treatment of these ADCs on
alloreactive T cell responses during an ongoing allogeneic response or prior to initiating an allogeneic response in
mice. These experiments establish that targeting this payload via the CD45 antibody preferentially killed actively
cycling allogeneic T cells and rendered residual T cells after ADC treatment incapable of mounting an allogeneic
response. These results establish that CD45–ADC mediates targeted elimination and neutralization of CD45+
effector cells. In the PGIA model, therapeutic intervention (day 11 post 3rd immunization) with CD45-ADC and BMT
halted disease progression, similar to the clinically-validated approach of multiple doses of anti-TNFα therapy.
These data show that CD45-ADC conditioning followed by congenic transplant is effective at immune reset and
shows comparable efficacy to clinically validated therapies.
We also generated novel anti-human CD45 ADCs and evaluated these for the ability to deplete hematopoietic and
immune cells in vitro and in vivo in humanized NSG mice. The CD45-ADC showed efficient killing of human
peripheral CD3+ cells from healthy donor (EC50 7.6 x10-10 M) and MS patients (EC50 1.5 x 10-10 M). In vivo, in
hNSG, single doses of the CD45-ADC was well-tolerated and led to substantial depletion of both lymphocytes and
hematopoietic stem cells (HSCs). A single dose CD45-ADC, but not Iso-ADC, resulted in elimination of disease-
causing T cells and reversal of clinical symptoms in a scleroderma-like chronic xenoGVHD model.
These results suggest that targeted immune depletion with a single treatment of Magenta CD45-ADC may be
sufficient for auto-HSCT and allow immune reset and re-establishment of immune tolerance. Targeted CD45-ADCs
may represent a safer and better tolerated approach for conditioning patients prior to immune reset through
autoHSCT and may significantly reduce the side effects associated with current conditioning.
A.
B. C.
d0 d1
MOG
PTX
d5
PTX Tx + BMT
d7
0
25
50
75
100
%C
D4
5.1
0
25
50
75
100
%C
D45
.1 O
F C
D11
b+
0 7 14 21 280
1
2
3
4
Days Post Immunization
EA
E C
lin
ical S
co
re
Tx
MOG
BMT
FTY Tx
FTY-720 3 mg/kg QD
CD45-ADC + BMT
Iso-ADC + BMT
Naked CD45 mAb (3 mg/kg 1x)
Vehicle + BMT
0
1000
2000
3000
4000
Rela
tive IL
-17A
+ T
cell #
D.
B6 (CD45.2+)
Figure 1. Participant Milestones.Milestones for each participant assigned to undergo myeloablative hematopoietic stem-cell transplantation (Panel A) or to receive cyclophosphamide (Panel B) are depicted from the time of informed consent. Organ failure refers to respiratory, renal, or cardiac failure. For early withdrawals from the trial, death was investigated with the use of site and public records. In Panels A and B, each black hash mark represents a clinical evaluation with pulmonary-function tests at the transplantation center. In Panel B, the asterisk identifies a participant who gave consent more than 12 months before randomization. The vertical dashed line at 54 months indicates participant status at the time of the primary end point
Sullivan, et al. N Engl J Med 2018; 378:35-47DOI: 10.1056/NEJMoa17033278
Efficacy of autoHSCT in MS Efficacy of autoHSCT in SSC
Muraro, et al. Nat. Rev. Neurology 2017; 13:391-405doi:10.1038/nrneurol.2017.81
Figure 1. Immune Reset via Autologous Hematopoietic Stem Cell Transplant is a Clinically-Validated,
Highly Effective Therapy in Multiple Autoimmune Diseases
MAGENTA PLATFORM ENABLES GENERATION OF
TARGETED ANTIBODY-DRUG CONJUGATES (ADCs) WITH
CUSTOMIZABLE PROFILES
Target:
Human CD45
Immune and HSC depletion
Payload
Engineering:
Modified human Fc enables rapid
clearance to enable transplant
Payload:
Kills Quiescent and
cycling cells
EXPERIMENTAL METHODS
Transplant
Cyclophosphamide
AN ANTI-HUMAN CD45-ADC ELIMINATES EFFECTOR CELLS IN
VITRO AND IN VIVO AND REDUCES DISEASE IN A
SCLERODERMA-LIKE MODEL OF XENOGVHD
14 Days Post ADC
Pre ADC
Iso-ADC CD45-ADC
Figure 5: Treatment with a single dose of an anti-human CD45 ADC results in elimination of human effector cells and
amelioration of disease. (A) A single dose off anti-human CD45-ADC was well tolerated and eliminated human hematopoietic
cells in the periphery (A) and progenitor cells in BM (B). Humanized NSG mice that had developed a chronic, xenoGVHD with skin
involvement after adoptive transfer of human PBMC were given a single dose of either an isotype ADC or an anti-human CD45-
targeting ADC. Images of selected animals show clear resolution of skin pathology and regrowth of hair in animals administered
CD45-ADC at 14 days post treatment, while isotype-ADC treated animals did not improve (C). Clinical scores for the animals
based on the size and presentation of skin lesions are summarized in (D); the top scoring animals in the isotype-ADC treated
group had to be euthanized at day 14 due to their symptoms. Durable depletion of peripheral human T cells was achieved in
animals treated with the anti-human CD45 ADC (E).
0
10
20
30
% h
CD
45
+ C
D3
+ (
of
Sin
gle
Ce
lls
)
Day 0
Day 7
Day 14
Day 62
-5 20 45 70
1
2
3
4
Days Post-Administration
Cli
nic
al
GV
HD
Sc
ore
CD45-ADC
Iso-ADC
Clinical Scores T Cell Depletion
Iso-ADC CD45-ADC
Group Mean Time toEAE onset
Incidence
Vehicle + BMT 11 100%Naked CD45 mAb 11 100%FTY-720 12 100%Isotype-ADC + BMT 28 42%CD45-ADC + BMT 38 25%
Donor Chimerism Myeloid chimerism
Clinical Scores
Splenic IL-17A+ T cells
Anti-Mouse CD45-ADC Anti-Human 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
Anti-mouse CD45-ADC
Anti-human CD45-ADC
Figure 3: Anti-CD45-ADC preferentially kills proliferating allogeneic T cells in vivo and renders quiescent T cells
incapable of mounting an allogeneic response. Host animals (CD45.1+) were treated with a single intravenous dose (3
mg/kg) administration of anti-mouse CD45-ADC, or isotype-ADC 7 days post initiation of scGvHD (CD45.2+ cells) (A&B).
Animals were euthanized on day 12 post initiation (5 days post ADC administration). Tissues (blood, spleen) were analyzed
for allogenic cells, T-cells, and proliferating T cells. A) Flow cytometric plots. B) Greater depletion of T-cells was observed
with CD45-ADC treatment in spleen or peripheral blood compared to Isotype-ADC. 7 days after exposure to 3 mg/kg of either
Isotype ADC or CD45-ADC, equal numbers of residual B6 T cells (H-2b) were labelled with CellTrace Violet adoptively
transferred into naïve, immunodeficient NSG hosts (H-2g7, H-2d). Cells were isolated from peripheral blood (d2 and d7) and
spleen (d7) post transfer and assessed for CTV staining intensity (C) and absolute number (D).
Ki-67TCRβ
Iso-ADC
CD45-ADC
CD
3
A. Spleen B.
ANTI-MOUSE CD45-ADC PREFERENTIALLY KILLS
PROLIFERATING T CELLS AND RENDERS QUIESCENT T CELLS
INCAPABLE OF GENERATING AN ALLOGENEIC RESPONSE
0
5000
10000
15000
20000
T cells on day 7 post transfer
# C
D45.2
+C
D3
+ c
ells
Peripheral Blood d7
Peripheral Blood d2
CellTrace Violet
Spleen d7
Peripheral bloodSpleen
Untreated
Isotype-ADC 3 mg/kg
CD45-ADC 3 mg/kg
Untreated
Isotype-ADC 3 mg/kg
CD45-ADC 3 mg/kg
0 2 4 6 80
500
1000
1500
2000
Days post transfer
# C
D45.2
+C
D3
+ c
ells
Untreated
Isotype-ADC 3 mg/kg
CD45-ADC 3 mg/kg
C. D.
Anti-mouse CD45-ADC
0
50000
100000
150000
200000
# host IFN+ spleen
Rela
tive IF
N
+ T
cell # Vehicle
Naked CD45 mAb
Isotype-ADC 3 mg/kg
CD45-ADC 3 mg/kg
FTY-720 d10
IMMUNE RESET AFTER ANTI-MOUSE CD45-ADC CONDITIONING
AND BMT AMELIORATES INFLAMMATORY ARTHRITIS
Figure 4: Therapeutic Treatment with CD45-ADC Enables Immune Reset via Congenic BMT and results in halt of
disease progression in a murine model of Rheumatoid Arthritis. BALB/c mice (CD45.2+) were given 3 immunizations
(study day 0, 21, and 42) with recombint human core G1 aggrecan (60 μg in 2 mg DDA) (A). Animals were treated on day 11
post the treated with a neutralizing monoclanonal antibody to murine TNFα received 500 μg/mouse IP weekly starting on
study day final immunization (study day 53) and conditioned animals were transplanted with Balb/c CD45.1+ congenic BM 48
hours later. Animals 53. Treatment with 2 mg/kg of CD45-ADC, but not Isotype-ADC, enabled full congenic donor chimerism
in peripheral blood (B) at 3 weeks post transplant. The clinical scoring system for the model as developed by Glantt and
Mikecz (Methods in Molecular Medicine, Vol. 102 (17):313-338. is shown in (D), with examples from control and CD45-ADC –
treated animals. Scores for the treatment groups over time are graphed in (E).
A.
B.
C.
-10 -5 0 5 10 15 20 25 30 350
4
8
12
Day Post Final Immunization
Cu
mm
ula
tive A
rth
riti
s S
co
re
TxBMT
***
TNF
BALB/c (CD45.2+)
rhG1A (60μg) in DDA adjuvant
d0 d42d21
Txd53
BMT (CD45.1+)
d55
Control
CD45-ADC
0
20
40
60
80
% C
D45.1
+(o
f to
tal C
D45
+)
0
20
40
60
80
100
% C
D45.1
+ (
tota
l G
r1+)
0
20
40
60
80
100
% C
D45.1
+ (
of
tota
l B
220
+)
0
20
40
60
80
100
% C
D45.1
+ (
of
tota
l C
D3
+)
Total Chimerism B Cell Chimerism T Cell ChimerismNeutrophil Chimerism
D.
-10 -5 0 5 10 15 20 25 30 350
4
8
12
Day Post Final Immunization
Cu
mm
ula
tive A
rth
riti
s S
co
re
TxBMT
anti-TNFa 500 g, QW
Naked CD45 mAb (3 mg/kg 1x)
Iso-ADC (2 mg/kg 1x)
CD45-ADC (2 mg/kg 1x)
***
TNF
CD45.1+
10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6
1000
10000
100000
1000000
Concentration (M)
Lu
min
esen
ce (
RL
U)
Human CD34+ CD38neg in Bone MarrowHuman β2M+ in Peripheral Blood
In vitro killing of T cells
B.A.
C. D. E.
E. F.
Anti-human CD45-ADC
10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6
1000
10000
100000
1000000
Concentration (M)
Lu
min
esen
ce (
RL
U)
Healthy donor
Isotype Control
MS donor #1
MS donor #2
MS donor #3
0
5000
10000
15000
20000
0
2000
4000
6000
8000
0
20
40
60
80
100
0
20
40
60
80
100
0
2 0
4 0
6 0
8 0
b lo o d p e rc e n ta g e s
% p
are
nt
Is o ty p e P B D 3 .0 m g /k g P B
1 0 4 -P B D 3 .0 m g /k g P B
Iso-ADC
CD45-ADC
Total
T cells
Spleen
% o
f C
D45.2
+
Ki-67+
T cells
#
CD
45.2
+
Total
T cells
Ki-67+
T cells
Total
T cells
Ki-67+
T cells
Total
T cellsKi-67+
T cells
#
CD
45.2
+
% o
f C
D45.2
+
Peripheral blood
0
25000
50000
75000
100000
# o
f C
D45
.1+
HS
C/f
em
ur
PBS
TBI
Naked CD45 mAb
Iso-ADC 2 mg/kg
CD45-ADC 2 mg/kg
TNF
0
25
50
75
100
150
% H
um
an
2M
+ (
of
Ba
se
line
)
Day 0Day 7
Day 14
0
5×106
1×107
1.5×107
2×107
2.5×107
Hu
ma
n
2M
+ C
ell
s/F
em
ur
0
50000
100000
150000
CD
34
+C
D3
8- C
ell
s/
Fe
mu
r
Human β2M+ in Bone Marrow
Iso-ADC CD45-ADC
PBS 3 1 3 mg/kg
Iso-ADC CD45-ADC
PBS 3 1 3 mg/kg
Iso-ADC CD45-ADC
PBS 3 1 3 mg/kg
Anti-mouse CD45-ADC