The Preclinical Development and Validation of a PD-L1 Antagonist in Mouse

Affimer Biotherapeutics: The preclinical development and validation of a PD-L1 antagonist in mouse

NGPT San Diego, 6th June 2017

Amrik Basran Chief Scientific Officer Avacta Life Sciences

• Avacta Life Sciences (AIM listed) established in 2012 to exploit Affimer IP • Sites in Cambridge (~23 staff) and Wetherby (~40 staff) • Raised £22m ($34m) in July 2015 for Affimer biotherapeutics with a focus on immuno-oncology and immuno- inflammation • Research collaboration and license deal with Moderna Therapeutics

2 Therapeutic Protein Scaffolds

VH CH1 • Most successful class of protein therapeutics VL CL fAb ScFv • But IgGs are large and limited routes of CH2 48 kDa 24 kDa administration C 3 H • Difficult manufacturing/disulphides/fragment stability IgG V dAb V dAb 150 kDa H L 12 kDa IgG based scaffolds • Smaller size • Mono- or multivalency • +/- Fc effector function • Microbial manufacturing options Anticalins DARPins Adnectins • Can be delivered by different routes of 3 Non- IgG based scaffolds administration (e.g. topical) Affimer Technology

• Based on Stefin A, a human intracellular protein

• 1/10th size of a mAb

• No disulphide bonds or post translational modifications

• Expressed at high levels

• We have freedom to operate

• Engineered to create large Affimer libraries (1x1010)

• Utilise phage display to identify binders 4 Library Generation: Phage Display

Affimer Gene Loop 4 Loop 2 Protein “displayed” Loop 2 Loop 4 on the tip of the virus 9 aa 9 aa

Affimer library containing over 10 billion different gene Microbial host sequences is then (E. coli) packaged with viral DNA

DNA encoding the Affimer gene and the virus. Affimer gene and 5 protein now “linked” Lead Identification: Phage Selections

Selection Pressure

Wash Binding Step Step

Target Antigen

Repeat Acid elution DNA of the phage

Infect and +Antigen -Antigen amplify in E. coli The Process: Lead Characterisation

~5-7 weeks Expression ELISA BIAcore Antigen Screening: Assay Phage Screening Sub-clone DNA SEC-MALLS biotinylation BIAcore Development (cross reactivity) binders Sequencing Solubility and QC ELISA etc Tm Cell assay Cross reactivity

Affinity Maturation

Lead Clones

Formatting Immunogenicity testing Developability assessment PK & efficacy 7 Immuno-oncology Strategy Combination Therapies and Agonists

T-cell Recruitment CAR-T T-cell

Tumour Drug Conjugates

Intratumoral Expression 8 Pharmacokinetics

100

Therapeutic window

10 %ID/mlSerum 1

Short serum half-life ~0.5hrs, due to renal clearance (~<60kDa) - acute indications - in vivo imaging reagents 0.1 0 5 10 15 20 25 30 Time (h) 9 Serum Half-life Extension Technologies

-S-

Human Serum Fc Fusions PEGylation Albumin

Utilising IgG-FcRn recycling Increased hydrodynamic size Affimer biotherapeutic binds to maintain high serum of the protein to prevent to HuSA in the circulation levels clearance via the kidneys

10 PD-L1 Program Immune Checkpoint Inhibitors: PD-L1

• PD-L1 plays a major role in immune suppression • Tumour cells that express PD-L1 on their surface appear “normal” and therefore invisible to the immune system • Blockade of the PD-L1/T-cell (PD-1) interaction reactivates the immune system • Numerous immune check-point proteins are now being targeted • Multiple anti-PD-1 and PD-L1 mAbs are in clinical development/approved

• Hundreds of clinical trials with PD-1/PD-L1 blockade and combination therapies Ott, et al., Clinical Cancer Research, 2013

12 Anti-PD-L1 Binders: Production in E. coli

• Identified a range of unique sequences • Ni-NTA purified (>95%) and expression levels ~200-350 mg/L at 15 ml scale

13 • Affimer binders compete for human PD-1/CD80 epitopes on PD-L1 Multimer Formatting: PoC With PDL1-141

14 Fc Formatting of PDL1-251 PDL1-251 Fc SEC-HPLC • PDL1-251 Fc Formatted as IgG1 Fc fusion PDL1-251 and expressed transiently in Expi293F cells • Purified using PrA sepharose followed by prep-SEC (yield

~200 mg/L) PDL1-251 Fc Biacore

• PDL1-251 Fc KD of ~40 pM by Biacore KD= ~40 pM

15 15 PD-1/PD-L1 Cell Based Assay

• Engineered Jurkat cell based signalling assay involving binding between two cells (Promega)

• PDL1-251 monomer has an EC50 ~1.1 μM

6 • PDL1-251 Fc has an EC50 ~40-50

nM (~25 fold improvement with n

o i t 4 formatting) c

u mAb 29E.2A3

n i

f PDL1-251 Fc

• Lead Affimers binders are now o d

l 2

o PDL1-251

undergoing affinity maturation, F linker optimisation etc 0 0.01 0.1 1 10 100 1000 10000 16 nM Mouse PD-L1 Program

mPD-L1 Biacore

• Human PD-L1 Affimer App KD = 316 pM antagonists do not bind mouse antigen • Initiated a mouse surrogate program for validation work mPD-L1 Competition ELISA • Affimer phage selections identified a potent tool molecule, PDL1-182 • Molecule is a competitive

inhibitor of mouse PD-1 IC50 = 20 nM

17 PDL1-182 Fc Production

• Formatted PDL1-182 as a human IgG1 Fc fusion (182 Fc1) • Expressed transiently in Expi293F cells

• Purified by Pr-A affinity 182 Fc1 SEC-HPLC chromatography followed by preparative SEC • Final purified yield >100mg/L yield, purity >95% (SEC-HPLC) > 95% purity

18 Characterisation of 182 Fc1 (I)

• Formatting of the Affimer protein significantly increase

binding affinity KD = 36 pM • Improvements most likely due to avidity effects

• Biacore binding improved ) 1 5 0

m A n ti m u P D -L 1 (1 0 F 9 .G 2 )

)

6 -

0 1 8 2 F c 1 5

~10 fold 4

(

o 1 0 0

b 182 Fc1

• i

Competition against PD-1 m

h )

0 EC 178pM 3

n 50

0 5

4 5 0

increased ~100 fold

(

0 1 0 0 .0 0 0 0 0 1 0 .0 0 0 1 0 .0 1 1 1 0 0 1 0 0 0 0 n M 19 Characterisation of 182 Fc1 (II)

• No functional mouse PD-L1 cell assay is available • Binding of 182 Fc1 to mouse cells was confirmed using flow cytometry before progressing to in vivo work

20 Pharmacokinetics of 182 P K p a rFc1a m e t e r s o f a s in g le d o s e e s c a la t io n

o f 1 8 2 F c 1 in m ic e )

L 1 0 0 0

m /

g 5 m g /K g 

( 1 0 0

• 182 Fc1 given as single 1 0 m g /K g

/ml)

o i g 2 0 m g /K g

bolus IV injection at 5,10 t μ

a 1 0

r t

and 20 mg/kg n

e 1

c n

• 3 animals per time point o

[182 Fc1] ( Fc1] [182 C

0 .1

m u • r Followed PK out to 7 days e 0 .0 1

S 0 5 0 1 0 0 1 5 0 2 0 0 • 182 Fc1 well tolerated T im e (h ) with no adverse effects Dose (mg/kg) Half-life (h) 5 20.9±1.3 10 19.2

21 20 59.9±5.3 CT26 Syngeneic Tumour Model

• Syngeneic mouse model utilizes immunocompetent mice bearing tumours derived from the strain of origin. • 5 groups with 10 animals per group (Balb/c) • Positive control 10F9.G2 (rat anti-mPD-L1 mAb) • Dosing each protein at 10 mg/kg every other day via IP route

Grp 1 = PBS Grp 2 = hFc1 Grp 3 = 182 Fc1 Grp 4 = 10F9G2 Grp 5 = rat IgG2b

22 DR= Day of randomization. 50 out of 70 tumours reached a mean volume of 91 ± 22 mm3 CT26 Syngeneic Model: Results

• Moderate anti-tumor effect seen with both 10F9G2 and 182 Fc1 Affimer • No macroscopic sign of toxicity or disease dissemination was recorded at the autopsy of mice • No significant body weight difference between groups • Repeat high dosing of 182 Fc1 was well tolerated

23 **** p<0.0001, α=0.05, multiple comparison 2-ways ANOVA Immunogenicity Testing Affimer Scaffold Immunogenicity Testing

• Therapeutic proteins have the potential to induce an immune response in vivo and generate anti-drug antibodies (ADA) • ADA can affect the PK and efficacy of the biological drugs by: Increasing rates of clearance Neutralising the molecule Potentially inducing adverse events • Several stages in assessing the immunogenicity of biologics:

In silico (identify T-cell epitopes) In vitro T-cell assays (e.g. human PBMCs, DC:T-cells) Humanised mice models

25 Immunogenicity Assessment: Human PBMC Assay Test therapeutic protein e.g. Affimer

Collect immune cells from human blood 50 μg/ml

Analyse immune cell 50 healthy donors activation and representing a broad Incubate for 1 week proliferation by flow population mix cytometry

26 Human PBMC Testing Results • In silico immunogenicity of the Affimer scaffold was determined Positive Responses to be low 50 100

40 80 s

• Affimer scaffold immunogenicity s 30 60

r r

20 40 o

o n

compared to Avastin (50 μg/ml) n 20 40

D e

• KLH positive control e 15 30

i s

s 10 20 o

• Positive response: SI>2 with o

P #

p<0.05 5 10 % • Core Affimer scaffold has a low 0 0 immunogenicity potential • Will be repeated on lead

molecules27 Summary

• Affimer therapeutics are an alternative to therapeutic antibodies with key benefits: o Generation of single digit/double digit nM binders from naïve libraries o Easily formatted e.g. multimers and Fc fusions with high expression levels o The Affimer scaffold is well tolerated in vivo with repeated high dosing • 182 Fc1 demonstrated a statistically significant moderate anti-tumour effect in the CT-26 syngeneic model, slowing tumour growth

• The parental scaffold shows a “low” immunogenicity risk comparable to a therapeutic mAb in human PBMC assays

• We have demonstrated that the Affimer technology has the properties necessary to generate therapeutic drugs

28 Acknowledgements

Avacta Life Sciences University of Leeds (BSTG)

• Emma Jenkins • Christina Rauber • Anna Tang • Estelle Adam • Lindsay McMorran • Mike McPherson • Flo Laurent • Graham Spence • Darren Tomlinson • Marine De Jaeger • Paul Shadbolt • Dino Ossola • Rob Ford • Ming Zhou • Andrew Wilcox • Jyrki Sivula • Matt Johnson The PD-L1 project has been • Emma Stanley supported by an Innovate UK Grant • Michele Writer • Lemy Tsikna