Presented at the 29th annual congress of the European Academy of Dermatology and Venereology (EADV) virtual from October 29‑31, 2020

INTRODUCTIONBruton Tyrosine Kinase• Bruton tyrosine kinase (BTK) is a promising target in immunology because of its expression in

B cells and innate immune cells, providing essential downstream signaling for B‑cell receptors, Fc receptors, and other innate cell pathways (Figure 1)1,2

Figure 1. BTK Plays a Critical Role in Innate and Adaptive Immunity

Megakaryocyte

PlateletsBTK

MicrogliaBTK

Natural killer cellBTK

T CellB cellBTK

Plasma cell

Mast cellBTK

BasophilBTK

NeutrophilBTK

EosinophilBTK

MacrophageBTK

MonocyteBTK

BTK Expression, functional inhibitionBTK Expression, no/limited functional effects

INNATE IMMUNITY ADAPTIVE IMMUNITY

• The role of innate immune cells is underappreciated in immune‑mediated dermatological diseases (Figure 2)3,4

- In lesional skin, innate cells such as mast cells, eosinophils, and neutrophils activate and accumulate, often correlating with tissue damage and disease severity - These skin‑resident and infiltrating immune cells can be targeted topically or systemically

Figure 2. Multiple BTK‑Dependent Cells Are Active in Skin Inflammation3,4

Dendritic cell

Natural killer cell

T cellB cell

T cell Langerhans cell

Plasma cellMast cell

Basophil

Neutrophil Eosinophil

Macrophage

Epidermis

Dermis

Keratinocytes

Topical BTK Inhibitor PRN473• PRN473 is a topically administered covalent inhibitor of BTK designed with Tailored Covalency®

to show durable, reversible BTK occupancy (Figure 3)5,6

• The longer BTK residence time optimizes prolonged, localized efficacy in rats and mice with low systemic exposure5

• Mechanistically, PRN473 inhibits IgE (FcεR)‑mediated activation of mast cells and basophils, IgG (FcγR)‑mediated activation of monocytes, and neutrophil migration7

• PRN473 was also demonstrated to be efficacious with excellent tolerability when given orally in the treatment of canine pemphigus foliaceus8

Figure 3. PRN473 Tailored Covalency®

COVALENTBINDING SITE

TARGETSITE

OFF‑TARGETSITE

NON‑COVALENTBINDING REGION

COVALENTBINDING REGION

MATERIALS AND METHODSIn Vitro Selectivity and Functional Assays• PRN473 1 µM was tested against a panel of 230 kinase targets in the KINOMEscan™ panel• Biochemical studies were performed to characterize the potency, selectivity, biochemical

on‑rate and off‑rate, and reversibility of the interaction between PRN473 and BTK• Selective inhibition of BTK function and occupancy and off‑target effects were evaluated in

cell‑based assays and human whole blood (HWB)

In Vivo PRN473 Treatment• PRN473 was applied topically at single and multiday doses of 0.25%‑4% before IgG/IgE

challenges• Positive controls were topical betamethasone, oral prednisolone, and antihistamines• Vehicle control was used as a comparator (t‑test with P < 0.05)

IgG‑Mediated Passive Rat Arthus Model9

• The passive Arthus reaction is an acute IgG antibody challenge model dependent on FcγR activation in macrophages and neutrophils

• Female Sprague‑Dawley rats (n = 5) were administered topical gel application with vehicle control; betamethasone diproprionate 0.05% (positive control); or PRN473 doses of 0.25%, 0.5%, 1%, or 2% for 3 or 16 h before IgG‑mediated skin challenge - Single‑day or multiday (3 days) evaluations were performed

• Inflammatory reactions were measured by reduction in intradermal extravasation diameter and optical dye density at OD610nm of Evans blue dye assessed after the challenge

IgE‑Mediated Passive Cutaneous Anaphylaxis (PCA) Mouse Model9

• The PCA model is an IgE FcεR‑mediated model, with mechanisms similar to those observed in human allergic disease - Measures IgE‑dependent responses via rapid mast cell activation, degranulation, and release of inflammatory mediators

• Female BALB/c mice (n = 5/group) were administered a single topical gel application 3 h before IgE‑mediated skin challenge with vehicle control, bethamethasone diproprionate 0.05% and diphenhydramine (positive control), or PRN473 doses of 0.5%, 1%, 2%, or 4%

• Thirty minutes after challenge, mice were euthanized, and skin samples were collected to assess the density of dye extravasation in the skin

Systemic Pharmacology: BTK Occupancy• Spleens were collected terminally and evaluated after the challenge to assess systemic effects

RESULTSPreclinical Selectivity and Functionality of PRN473• PRN473 exhibited selectivity to 6 of 230 kinases with >90% inhibition at 1 µM (Figure 4 left

panel) and durable in vitro occupancy with BTK, TXK, TEC, and BMX, but not BLK and ITK (Figure 4 right panel)

Figure 4. Preclinical Optimization of PRN473 for Selectivity, Potency, and Durability

Selectivity Profile (Kinase Panel) Durable OccupancyOver Time

0

20

40

60

80

100

1 6 24

Occ

upan

cy In

Vitr

o, %

Time, h

Kinase Assay

PRN473IC50 ± SD, nM

1.8 ± 0.2

3.4 ± 1.0

2.0 ± 0.2

1.8 ± 0.3

2.2 ± 0.6

300 ± 10

22 ± 2.0

>150

>370

BTKTXKTECBMXBLKITK

ERB‑B4

FGR, FYN, LCK, LYN, SRC, YESEGFR, ERB‑B2

BTKTXKTECBMXBLKITK

Kinase selectivity profile was evaluated for PRN473 against 230 kinases; dots represent individual kinases with 6 out of 230 panel showing >90% inhibition at 1 µM.BLK, B‑cell lymphocyte kinase; BMX, bone marrow tyrosine kinase on chromosome X; BTK, Bruton tyrosine kinase; ERB family members: ERB‑B4, EGFR, and ERB‑B2; ITK, interleukin‑2‑inducible T‑cell kinase; SRC family members: BLK, YES, LCK, FGR, FYN, SRC, and LYN; TEC, tyrosine protein kinase (family members BMX, TXK); TXK, nonreceptor tyrosine kinase of the TEC family.

IgE‑Mediated PCA Mouse Reaction• Significant dose‑dependent inhibition of mouse PCA IgE‑mediated reaction was also observed with single and multiday

applications of PRN473 ≥1%, approaching positive controls with higher strengths up to 4% PRN473 (Figure 6)• Multiday (3 days) treatment achieved similar levels of inhibition with respect to single administration (data not shown)

Figure 6. Dose‑Dependent Inhibition in IgE Antibody‑Mediated Mouse Passive Cutaneous Anaphylaxis (PCA) With Single Application of PRN473

Dye

Den

sity

(OD

610)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 3h before IgE

No treatmentGel Vehicle

BetamethasoneDiphenhydramine

4% PRN4732% PRN473

1% PRN4730.5% PRN473

**

**

**

**

**

**P < 0.001 versus vehicle.

Systemic Pharmacology• Systemic BTK occupancy evaluated with topical PRN473 in Arthus rat and PCA mouse models resulted in low to no

systemic accumulation, suggesting that localized exposure to PRN473 provided the major role for the efficacy (data not shown) - No measurable systemic BTK occupancy in the Arthus rat model with single or multiday doses of topical PRN473 2% or with single application of PRN473 2%‑4% in PCA mouse model - <50% systemic BTK occupancy with multiday topical PRN473 2%‑4% in the PCA mouse model

CONCLUSIONS

• Preclinical studies of PRN473 provide a strong biological basis for targeting skin innate immune cell responses with a rapid onset of action with once‑daily topical administration and minimal systemic exposure

• PRN473 effectively inhibited IgG (FcγR)‑ and IgE (FcεR)‑mediated signaling equivalent to topical or oral corticosteroids and prevented downstream immune effects locally in the skin

• Overall, dose‑dependent efficacy was demonstrated with topical PRN473 in preclinical models of immune‑mediated skin diseases, supporting future clinical studies

- Email for more information: clinicaltrials @ principiabio.com

REFERENCES1. Crofford LJ, et al. Exp Rev Clin Immunol. 2016:12:763‑773.2. Rip J, et al. Crit Rev Immunol. 2018:38:17‑62.3. Quaresma JAS. Clin Microbiol Rev. 2019;32:e00034.4. Richmond JM, Harris JE. Cold Spring Harb Perspect Med.

2014;4:a015339.

5. Bradshaw JM, et al. Nat Chem Biol. 2015;11:525‑531.6. Bisconte A, et al. J Immunol (AAI). 2015;194(1 suppl):139.6.7. Herter JM, et al. Br J Pharmacol. 2018;175:429‑439.8. Goodale EC, et al. Vet Dermatol. 2020;31:291‑e71.9. Chang BY, et al. Arthritis Res Ther. 2011;13:R115.

ACKNOWLEDGMENTSThese studies were sponsored by Principia Biopharma Inc, A Sanofi Company, South San Francisco, CA, USA. Editorial support was provided by Second City Science and funded by Principia Biopharma Inc, A Sanofi Company. The authors directed development of the presentation and are fully responsible for all content and editorial decisions

DISCLOSURESAll authors report employment with and stock ownership from Principia Biopharma Inc, A Sanofi Company.

CORRESPONDING AUTHORCopies of this poster are for personal use only and may not be reproduced without permission from the corresponding author of this posterCorresponding author email: Claire.langrish@principiabio.com

• In vitro, PRN473 inhibited B‑cell activation and FcR signaling in monocytes and mast cells, and in the absence of cytotoxic effects and limited functional effects in basophils, epidermal growth factor receptor (EGFR), and cells absent BTK signaling (Table 1A)

• PRN473 demonstrated durable BTK inhibition at 4 h that was partially sustained through 18 h (Table 1B)

Table 1. Preclinical Target Specificity and BTK Occupancy for PRN473

A. Cell‑Based Target Specificity Assays

On‑Target Activity IC50 ± SD, nM

BTK Ramos B cell occupancy 30 ± 15

FcγR IgG‑induced TNFα production in human monocytes 76 ± 40

FcεR IgE mast cell degranulation and β‑hexaminidase release 89

FcεR IgE mast cell degranulation and histamine release 175

BCR B cell activation in HWB 274 ± 95

BTK BTK occupancy in whole blood 364 ± 112

FcεR IgE‑induced basophil CD63 activation in HWB 1130 ± 510

Off‑Target Activity (>5000)

TCR T cell activation in whole blood >7900

N/A EGFR EGFR signaling reporter assay >5000

N/A Cytotoxicity HCT116 cell (no BTK) cytotoxicity >20,000

B. BTK Target Engagement – Occupancy in Human B CellsTime Ramos B cell BTK Occupancy ± SD

4 h 91% ± 3

18 h 54% ± 6

BCR, B‑cell receptor; BTK, Bruton tyrosine kinase; EGFR, epidermal growth factor receptor; HWB, human whole blood; Ig, immunoglobulin; TNFα, tumor necrosis factor alpha.

IgG‑Mediated Passive Rat Arthus Reaction• Significant dose‑dependent inhibition of IgG‑mediated passive Arthus reaction in rats was

observed with single and multiday topical PRN473 ≥0.5% strengths (Figure 5) - Equivalent inhibition to topical betamethasone was observed with 1% and 2% PRN473 - Inhibition was maintained when PRN473 was administered 16 h before challenge, reinforcing extended anti‑inflammatory activity with once‑daily application

Figure 5. Dose‑Dependent Inhibition in IgG Antibody‑Mediated Arthus Reaction Rat Model Single (A) and Multiday (B) Application of PRN473

16h before IgG

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Dye

Den

sity

(OD

610)

3h before IgG

Dye

Den

sity

(OD

610)

16 h before IgG

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 3h before IgG

Gel Vehicle Betamethasone2% PRN473 1% PRN4730.5% PRN473 0.25% PRN473

** **** **

** ** ****

** **

* **

**** **

*

A. Single Topical Application

B. Multiday (3 days) Topical Applications

*P < 0.05, **P < 0.001 versus vehicle.

Mechanisms of Topical Bruton Tyrosine Kinase Inhibitor PRN473 in Immune‑Mediated Models of Skin Disease

Yan Xing, Katherine A. Chu, Jyoti Wadhwa, Wei Chen, Jiang Zhu, Jin Shu, Matthew C. Foulke, Natalie Loewenstein, Philip Nunn, Kolbot By, Pasit Phiasivongsa, David M. Goldstein, and Claire L. Langrish

Principia Biopharma Inc, A Sanofi Company, South San Francisco, CA, USA

E‑Poster P1572