#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) | Copies of this poster are for personal, noncommercial use only and are not to be published in any form | All authors are employees of Monte Rosa Therapeutics |
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Monte Rosa Therapeutics Inc. published this content on 21 May 2024 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 21 May 2024 16:42:06 UTC.