Monte Rosa Therapeutics : EULAR 2024 – MRT 6160, a VAV1 Directed Molecular Glue Degrader, Reduces Joint Inflammation and Autoantibody Production in a Collagen Induced Arthritis Autoimmune Disease Model

#POS1200: MRT-6160, a VAV1-Directed Molecular Glue Degrader, Reduces Joint Inflammation and Autoantibody Production in a Collagen-Induced Arthritis Autoimmune Disease Model

Adam Cartwright2, Foram Desai1, Shailee Vora1, Lucas Gyger2, Xudong Wang1, Katie May1, Daniel Lam 1, Peter Trenh1, Xavi Lucas2, Mary Zlotosch1, Sophia Nguyen1, Elisa Liardo2, Daric Wible1,

Ilaria Lamberto1, Bradley Demarco1, Chris King1, Debora Bonenfant2, John Castle1, Markus Warmuth1, Sharon Townson1, Eswar Krishnan1, Filip Janku1, Laura McAllister2, Alison Paterson1, Marisa Peluso1

1Monte Rosa Therapeutics Inc., 321 Harrison Ave, Boston, MA 02118, United States

2Monte Rosa Therapeutics AG, WKL-136.3, Klybeckstrasse 191, 4057 Basel, Switzerland

VAV1 is a guanine nucleotide exchange factor with a critical role in T- and

B-cell receptor signaling and activity

T cell receptor VAV1 signaling	B cell receptor VAV1 signaling
pathway	pathway

•	VAV1 expression is highly restricted to immune cells
•	VAV1 is required for antigen receptor-mediated signaling of T- and B-cells
	CRISPR-mediated1 or genetic loss2 of VAV1 is associated with decreased effector

MRT-6160-induced degradation of VAV1 attenuates T cell activation and effector	Oral dosing of MRT-6160 at disease onset attenuates disease progression in a
functions	collagen-induced arthritis (CIA) autoimmune murine disease model

				CD69			120		Proliferation		(%)	160						TH subset cytokines			7	Vehicle			✱✱
	140
						(%)								120												IFNγ	TH1
	120						100							level												TNFα		6	Anti-TNF, 10 mg/kg		15	✱
(%)	100						Proliferation	80							cytokineRelative													IL-2				MRT-6160, 1 mg/kg	ScoreClinical(d21)
80													80												CXCL10		ScoreClinical SEM)±(mean	5	12
					60
CD69
+
	60							40																				IL-5	TH2					9
	40														40												IL-17A			4
																										TH17
								20
	20																										IL-22
	0							0								0				0.01	0.1		1	10	100	1000			3			6
	0.001	0.01	0.1	1	10	100	1000	0.001	0.01	0.1	1	10	100	1000		0.0001 0.001
			MRT-6160 (nM)					MRT-6160 (nM)										MRT-6160 (nM)					2			3
												TH17 Polarization																1			0
																											0
																DMSO)to	1.5													Days from treatment start		Vehicle
																1.0															MRT-6160, 1 mg/kg
																																-7-6-5-4-3-2-1 0 1 2 3	4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21		Anti-TNF, 10 mg/kg
																(normalized								Stimulated
																													DBA/1 mice were immunized with bovine collagen-II emulsified in complete Freund's adjuvant on day 0 (intravenously), then boosted with chicken collagen-II emulsified in incomplete Freund's adjuvant on
																	0.5												day 21 (subcutaneously). Mice were randomized into treatment groups following disease onset and treated with vehicle or MRT-6160 (PO QD) or anti-TNF (IP, TIW) for 21 days. Clinical scores (0-4) are
																												the sum of individual paws from blinded assessment of inflammation and ankylosis. Graphs show longitudinal clinical scores (mean ± SEM, left) and clinical scores on day 21 (first to third quartile with
																IL-17A														min/max, right). Statistical analysis was performed using a one-way ANOVA with Dunnett's comparison. *p<0.05, **p<0.01. N = 15 mice/group.
																0.0												MRT-6160 reduces serum levels of pro-inflammatory cytokines associated with
																			te	.0	0	3	3	0
																				d	3	3	0		0
																			la		0	.			3
																	ti												rheumatoid arthritis
																	u			MRT-6160 (nM)
																	ns	m
																U															✱✱✱
																																✱✱✱✱		✱✱✱

•

functions of both T and B cells

MRT-6160 is a rationally designed molecular glue degrader that selectively

degrades VAV1 in human and mouse immune cells

Upper row: Purified primary human pan-T cells were pre-treated with MRT-6160 for 24 hrs followed by ⍺CD3/⍺CD28 TCR stimulation and subsequent analyses by flow cytometry (CD69, proliferation) or MSD

(cytokines). N = 3 donors. Lower row: Purified primary human CD4+ T cells were treated with MRT-6160 for 24 hrs prior to polarization to a TH17 phenotype by ⍺CD3/⍺CD28 stimulation in the presence of IL-1β, IL- 6, IL-23, TGFβ, ⍺IFNɣ, and ⍺IL-4.IL-17A levels (pg/mL) in the supernatant were assessed by AlphaLISA after 3 days. N = 3 donors.

MRT-6160-induced degradation of VAV1 attenuates B cell activation and effector

functions

Plasmablast

(pg/mL)

ns

8

6

4

(pg/mL)

800

600

400

✱✱✱

✱

(pg/mL)

40

30

20

ns

(pg/mL)

10

8

6

ns

Molecular glue degraders (MGD) function to induce structural changes in ubiquitin ligases, such as cereblon, to drive the formation of ternary structures with a target protein.

Following binding of cereblon to the target, this protein is then ubiquitin tagged and subsequently degraded via the proteasome- mediated degradation machinery of the cell.

MGDs can induce degradation of otherwise 'undruggable' proteins as the mechanism does not require a classical binding pocket, contrary to conventional protein inhibitors, significantly increasing the target space and potential utility across a range of diseases.

Cereblon
	Ubiquitin
MGD, e.g.	chain
Ubiquitination
MRT-6160
Neosubstrate	Ternary
complex
e.g. VAV1
Cereblon	Proteasome-
mediated
+ MGD
degradation of
	neosubstrate

	CD69	IL-6		Soluble IgG		differentiation
	120	120	120			120
	100	100	100		(%)	100
(%)	80	80	80		80

IL-1β

2

0

IL-6

200

0

TNF

10

0

IL-17A

4

2

0

+	60						IL-(%)6	60					IgG(%)	60						Plasmablasts	60
CD69
40						40					40						40
20						20					20						20
	0							0						0							0
	0.001 0.01	0.1	1	10	100	1000		0.0010.01	0.1	1	10	100 1000		0.001 0.01	0.1	1	10	100	1000		0.0010.01	0.1	1	10	100 1000

MRT-6160 (nM)

MRT-6160 (nM)

MRT-6160 (nM)

MRT-6160 (nM)

Naive

Vehicle

Anti-TNF, 10 mg/kg

MRT-6160 , 1 mg/kg

Serum samples were collected from mice at the end of the study. Pro-inflammatory cytokine levels were assessed by Mesoscale Discovery U-PLEX platform as per the manufacturer's instructions. Graphs show quantification of cytokines (pg/mL) for each indicated cytokine in naïve, vehicle, anti-TNF, and MRT-6160 treatment groups. Naïve group was excluded from statistical analysis and shown for comparison to diseased groups. Statistical analysis was performed using a one-way ANOVA with Dunnett's multiple comparisons. ns = not significant, *p>0.05, ***p<0.001, ****p<0.0001. N = 15 mice/group (diseased) or 5 mice/group (naïve).

MRT-6160 reduces serum levels of T-cell-dependentanti-collagen II IgG1 and

total IgG antibodies

% of Vav1 degradation

Human PBMCs

150

100

50

0
-50
0.01	0.1	1	10	100	1000

MRT-6160 (nM)

MRT-6160 (nM)

DMSO

VAV1

β-actin

% of Vav1 degradation

Mouse Splenocytes

150

100

50

0
-50
0.01	0.1	1	10	100	1000

MRT-6160 (nM)

MRT-6160 (nM)

DMSO

VAV1

β-actin

Purified primary human B-cells were pre-treated with MRT-6160 for 24 hrs followed by stimulation with anti-IgM and IL-4 and analysis of CD69 expression and IL-6 secretion 24 hrs post stimulation or stimulation with anti-IgM, sCD40L, IL-21,IL-2, and BAFF and analysis of soluble IgG and plasmablast differentiation on day 5 post stimulation. Data are normalized to respective stimulation DMSO control. N = 3 donors.

Summary and Future Development

• MRT-6160 is a first-in-class VAV1 MGD that attenuates TCR- and BCR-mediated activity in vitro & in vivo.
• Degradation of VAV1 attenuates T/B cell activation, proliferation, effector functions, and differentiation.
• Therapeutic administration of MRT-6160-mediated degradation of VAV1 attenuates disease progression in
•	a collagen-induced arthritis disease model.
Degradation of VAV1 at disease onset attenuates serum pro-inflammatory cytokines and autoantibody
•	production .
Given the in vitro and in vivo MOA profile shown, MRT-6160 has strong potential to alleviate disease
	symptoms in multiple autoimmune and inflammatory diseases including rheumatoid arthritis, multiple

Anti-collagen II IgG1 (units/mL)

1.5×107

1.0×107

5.0×106

✱✱

✱

Anti-collagen II Total IgG (units/mL)

8×106

6×106

4×106

2×106

✱

ns

Vehicle

Anti-TNF, 10 mg/kg

MRT-6160, 1 mg/kg

Serum samples were collected from mice at the end of the study. Levels of anti-collagen II IgG1 and total IgG were

Human PBMCs and mouse splenocytes were treated overnight with dose-range of MRT-6160, after which VAV1 protein levels were assessed by JESS. Percentage (%) VAV1 degradation was calculated by normalizing VAV1 expression to β-actin loading control and shown as relative to DMSO control. Data from N = 3 biological replicates. Human PBMCs and mouse splenocytes were treated for 24 hrs with 10 μM MRT-6160 then assessed by quantitative tandem mass tag proteomics. The y-axis represents p-value[-log10]; the x-axis represents protein fold change [log2] relative to DMSO (0.1%) control samples. Dark blue circles represent CRBN neosubstrates including the target, VAV1, and other known cereblon neosubstrates; GSPT1, IKZF1, IKZF3, CSNK1A1 (CK1α), SALL4, and ZFP91. Purple circles represent VAV family members VAV2 and VAV3.

sclerosis, inflammatory bowel disease, and psoriasis, amongst others.

• MRT-6160 is a development candidate with IND submission upcoming.

0.0

0

assessed using an ELISA immunoassay. Graphs show serum levels (units/mL) of anti-collagen-II IgG1 (left) and total IgG (right). Statistical analysis was performed using a one-way ANOVA with Dunnett's multiple comparisons. ns = not significant, *p<0.05, **p<0.01. N = 15 mice/group.

References: 1. Schmidt et al. Science (2022); 2. Fujikawa et al. J. Exp. Med. (2003)

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All authors are employees of Monte Rosa Therapeutics