Monte Rosa Therapeutics : DDW 2024 – MRT 6160, a VAV1 Directed Molecular Glue Degrader, Inhibits Disease Progression in a T cell Transfer Mediated Colitis Model Concomitant with Reduced Calprotectin Expression

#Tu1727: MRT-6160, a VAV1-Directed Molecular Glue Degrader, Inhibits Disease Progression in a T-cell Transfer Mediated Colitis Model Concomitant with Reduced Calprotectin Expression

Adam N. R. Cartwright2, Shailee Vora1, Lucas Gyger2, Foram Desai1, Xudong Wang1, Katie May1, Sophia Nguyen1, Daniel Lam1, Peter Trenh1, Xavi Lucas2, Mary Zlotosch1, Elisa Liardo2, Daric Wible1, Ilaria Lamberto1, Bradley Demarco1, Chris King1, Debora Bonenfant2, Sharon Townson1, Steven De Beukelaer2, Eswar Krishnan1, John Castle2, Markus Warmuth1, Owen Wallace1, 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 critical guanine nucleotide exchange factor in T-cell receptor signaling and activity

MRT-6160 inhibits TCR activity and CD4+ TH17 polarization in a

concentration-dependent manner

MRT-6160 inhibits colitis disease progression, colon inflammation, and

mucosal cytokine levels in a T-cell transfer model of colitis

CD69 activation (%)

140

120

100

80

60

40

20

0

	CD69						Cytokines
				(%)	160
				level	120
				cytokine	80
				Relative
				40
					0
0.01	1	100	10000		0.0001 0.001	0.01	0.1	1	10	100	1000
	MRT-6160 (nM)					MRT-6160 (nM)

IFNγ

TNFα

IL-2 CXCL10

IL-5

IL-17AIL-22

Th1

Th2

Th17

Proliferation (%)

Proliferation

120

100

80

60

40

20

0

0.01

1

100

10000

MRT-6160 (nM)

			CD45RB	low	non-pathogenic control			✱✱✱✱
		6
		Vehicle
		5				120	✱✱✱✱
		Anti-TNF, 25 mg/kg
	(mean ± SEM)
DAI Score	4	MRT-6160, 1 mg/kg	(AUC, mean ± SEM)	90
			DAI Scores
3			60	59%
2
1			30	85%

• VAV1 expression is highly restricted to immune cells
• VAV1 is a pivotal scaffolding protein and signaling molecule downstream of the TCR
• CRISPR-mediatedKO1 or germline genetic loss2 of VAV1 is associated with decreased functions of T cells
• VAV1 degradation is predicted to impact T cell and B cell function and treat a broad set of autoimmune and inflammatory diseases

MRT-6160 is a rationally designed molecular glue

TH17 Polarization				IL-17A
	1500
(pg/mL)	1200
900
17A-IL
600
	300
	0
	0.0001	0.001	0.01	0.1	1	10	100	1000
				MRT-6160 (nM)

Purified primary human pan-T cells were pre-treated with MRT-6160 for 24 hrs followed by ⍺CD3/⍺CD28 TCR stimulation and analyses after 24 hrs (CD69, flow cytometry, upper left), 48 hrs (cytokines, MSD, upper center) or 96 hrs (proliferation, flow cytometry, upper right). Data are normalized to TCR-stimulated DMSO control. N = 5 donors. Purified primary human CD4+ cells were pre-treated with MRT-6160 for 24 hrs, followed by ⍺CD3/⍺CD28 TCR stimulation and polarization towards the TH17 subtype as indicated. After 3 days, IL-17A levels (pg/mL) in the supernatant were assessed by AlphaLISA (lower). Representative data from N = 2 donors.

MRT-6160 reduces expression of inflammatory disease-associated T cell differentiation and chemokines and calprotectin subunit genes

0															0
0	3	6	9	12	15	18	21	24	27	30	33	36	39	42	low
CD45RB non-pathogenic control
			Time post treatment initiation (Day)			Vehicle
					Anti-TNF, 25 mg/kg
															MRT-6160, 1 mg/kg

Splenic CD45RBhigh (pathogenic, 0.5 x 106 cells/mouse) and CD45RBlow (no disease control , 0.5 x 106 cells/mouse) were enriched from Balb/c donors by negative magnetic selection and FACS. Enriched cells were then transferred into SCID mice and treatment was initiated 2 hrs post-transfer. Mice were treated with vehicle (PO, QD), anti-TNF (IP, Q3D), or MRT-6160 (PO, QD) for 42 days. Disease activity index (DAI; determined by weight loss and stool consistency) were assessed every 3 days until the end of the study. Graphs show DAI throughout the study (upper left,) and DAI area under curve (AUC) with disease incidence inhibition (upper right). Colons were excised at study termination then measured for weight and length (lower far left). A section of the colon was taken for independent, blinded histopathological scoring by H&E staining (lower middle left) for crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell presence, and crypt abscess presence. Colon mucosa were assessed by cytokine bead array for levels of IL-6 and TNF. Graphs show concentrations (fg/mg) of IL-6 (lower middle right) and TNF (lower far right). Statistical analysis was performed using a one-way ANOVA with Dunnett's

degrader that selectively degrades VAV1

10-30

Clec4e

multiple comparisons (excluding CD45RBlow control group). ****p<0.0001. N = 8 recipient mice per group.

MRT-6160 reduces CD4+ T cell expression of TNF and IL-17A in

Cereblon

MGD, e.g.

Ubiquitin chain

Ubiquitination

10-20

mesenteric lymph nodes in a non-depleting manner

CD4+ T cell frequency

Treg cell frequency

TNF+ frequency

IL-17A+ frequency

MRT-6160

		Neosubstrate
		e.g. VAV1		Ternary
				complex
								Proteasome-mediated
Cereblon +								degradation of
MGD								neosubstrate

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

Following binding of cereblon to the target protein, this target 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 utility across a range of diseases.

p value	10-10
Adjusted
	100

S100a9 Cxcl5	Selenbp1
Mmp3
S100a8

			ns
	T cells		ns
		15
	Regulatory	)	10
	+
	CD4
low
CD45RB non-pathogenic control
	+	of
Vehicle	CD25	(%	5
Anti-TNF, 25 mg/kg
MRT-6160, 1 mg/kg	+
FoxP3
		0

Mesenteric lymph nodes were excised from mice at termination, homogenized into a single cell suspension, and stimulated with PMA/ionomycin with protein transport inhibitors for 6 hrs. Cells were then stained for surface and intracellular markers and assessed by flow cytometry. CD4+ frequencies were gated on viable CD45+CD3+ events and shown as a percentage of CD45+ cells. Regulatory T cell (FoxP3+CD25+) frequencies were gated on viable CD45+CD4+ events and shown as a percentage of CD4+ cells. Cytokine analysis was gated on viable CD45+CD3+CD4+CD69+ events. Statistical analysis was performed using a one-way ANOVA with Dunnett's multiple comparisons compared to vehicle treated groups; non-pathogenic control was excluded from statistical analysis. ns = not significant, **p<0.01, ****p<0.0001. N = 8 recipient mice per group.

Summary and Future Development

Human PBMCs	Mouse Splenocytes

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.

1010

-4

-2

0

2

4

Log2 fold change

Gene

L2FC

Adj. p-value

Function

Clec4e

-3.46

1.2 x 10-30

Induces Il1b expression and T 17 differentiation

H

S100a9

-3.40

3.6 x 10-9

Calprotectin subunit, induces inflammatory activation of endothelial cells

S100a8

-3.24

8.8 x 10-7

Calprotectin subunit, induces inflammatory activation of endothelial cells

Cxcl5

-2.90

7.6 x 10-10

Ligand for CXCR2, induces migration and chemotaxis of T cells

Mmp3

-2.88

9.7 x 10-10

Cleaves extracellular matrix proteins and activates pre-cursors of TNF⍺ and IL-1β

Selenbp1

2.11

1.7 x 10-10

Selenium-binding protein, associated with long-term remission in UC patients

Colons were excised from mice at termination and stored in RNAlater. RNA was extracted from colon samples and quality was assessed by NanoDrop and Qubit. Gene counts were performing using the NanoString Autoimmune Profiling Panel and processed according to the manufacturer's instructions. Background signal was determined at twice the SD above the mean of the negative control. Data were normalized to 8 housekeeping genes and median of the ratio was used. Graphs show differentially expressed genes (DEG) in MRT-6160 vs vehicle treated mice. Table shows log2 fold change, p-value, and description of labelled genes in volcano plot.

• MRT-6160is a novel, potent, and selective VAV1 molecular glue degrader, which inhibits TCR- induced activation, cytokine production, proliferation, and TH17 polarization.
• In a T-cell transfer model of colitis, MRT-6160 prevents disease progression, colon inflammation, and reduces pro-inflammatory cytokine production.
• Transcriptional analysis of colon tissue shows reduced expression of TH17-associated, chemokine, and calprotectin subunit genes.
• These data suggest that MRT-6160 has strong potential to alleviate disease symptoms in
  multiple T-cell and/or TH17 mediated autoimmune and inflammatory diseases including inflammatory bowel disease.
• MRT-6160is a development candidate currently in IND-enabling studies.

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