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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