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

Amrik Basran

Chief Scientific Officer

NGPT San Diego, 6th June 2017

Avacta Life Sciences

2

• 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

fAb48 kDa

ScFv24 kDa

VH dAb VL dAb

12 kDa

CH2

CH3

CH1VH

CL

VL

IgG150 kDa

Therapeutic Protein Scaffolds

DARPinsAnticalins Adnectins

• Most successful class of protein therapeutics

• But IgGs are large and limited routes of administration

• Difficult manufacturing/disulphides/fragment stability

• Smaller size

• Mono- or multivalency

• +/- Fc effector function

• Microbial manufacturing options

• Can be delivered by different routes of administration (e.g. topical)

IgG based scaffolds

Non- IgG based scaffolds3

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 binders4

Library Generation: Phage Display

Affimer library

containing over 10

billion different gene

sequences is then

packaged with viral

DNA

Microbial host(E. coli)

DNA encoding the Affimer gene and the virus. Affimer gene and

protein now “linked”

Protein “displayed” on the tip of the virus

Loop 4 Loop 2Affimer Gene

Loop 29 aa

Loop 49 aa

5

Lead Identification: Phage Selections

Acid elution of the phage

Infect and amplify in E.

coli

Target Antigen

Wash Step

Binding Step

Repeat

Selection Pressure

-Antigen+Antigen

DNA

The Process: Lead Characterisation

Antigen biotinylation

and QC

Phage Screening(cross reactivity)

Assay Development

Sub-clone binders

Screening:BIAcore

ELISA etc

DNA Sequencing

~5-7 weeksExpression

ELISABIAcore

SEC-MALLSSolubility

TmCell assay

Cross reactivity

Lead Clones

Affinity Maturation

7

Formatting Immunogenicity testing

Developability assessmentPK & efficacy

CAR-T

Immuno-oncology Strategy

8

Combination Therapies and Agonists

T-cell Recruitment

Drug Conjugates

T-cell

Tumour

Intratumoral Expression

9

Pharmacokinetics

Time (h)

%ID

/ml

Se

rum

0.1

1

10

100

0 5 10 15 20 25 30

Short serum half-life ~0.5hrs, due to renal clearance (~<60kDa)- acute indications- in vivo imaging reagents

Therapeutic window

Serum Half-life Extension Technologies

-S-

PEGylation

Utilising IgG-FcRn recycling to maintain high serum

levels

Increased hydrodynamic size of the protein to prevent clearance via the kidneys

Affimer biotherapeutic binds to HuSA in the circulation

10

Human Serum Albumin

Fc Fusions

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

12

Ott, et al., Clinical Cancer Research, 2013

• Identified a range of unique sequences

• Ni-NTA purified (>95%) and expression levels ~200-350 mg/L at 15 ml scale

• Affimer binders compete for human PD-1/CD80 epitopes on PD-L1

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

13

Multimer Formatting: PoC With PDL1-141

14

• Formatted as IgG1 Fc fusion and expressed transiently in Expi293F cells

• Purified using PrA sepharosefollowed by prep-SEC (yield ~200 mg/L)

• PDL1-251 Fc KD of ~40 pM by Biacore

15

Fc Formatting of PDL1-251PDL1-251 Fc SEC-HPLC

KD= ~40 pM

PDL1-251 Fc Biacore

15

PDL1-251 Fc

PDL1-251

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

• PDL1-251 monomer has an EC50

~1.1 μM

• PDL1-251 Fc has an EC50 ~40-50 nM (~25 fold improvement with formatting)

• Lead Affimers binders are now undergoing affinity maturation, linker optimisation etc

16

PD-1/PD-L1 Cell Based Assay

0.01 0.1 1 10 100 1000 100000

2

4

6

nM

Fo

ldo

fin

du

cti

on

mAb 29E.2A3

PDL1-251

PDL1-251 Fc

• Human PD-L1 Affimer antagonists do not bind mouse antigen

• Initiated a mouse surrogate program for validation work

• Affimer phage selections identified a potent tool molecule, PDL1-182

• Molecule is a competitive inhibitor of mouse PD-1

17

Mouse PD-L1 Program

App KD = 316 pM

IC50 = 20 nM

mPD-L1 Biacore

mPD-L1 Competition ELISA

• Formatted PDL1-182 as a human IgG1 Fc fusion (182 Fc1)

• Expressed transiently in Expi293F cells

• Purified by Pr-A affinity chromatography followed by preparative SEC

• Final purified yield >100mg/L yield, purity >95% (SEC-HPLC)

18

PDL1-182 Fc Production

182 Fc1 SEC-HPLC

> 95% purity

• Formatting of the Affimer protein significantly increase binding affinity

• Improvements most likely due to avidity effects

• Biacore binding improved ~10 fold

• Competition against PD-1 increased ~100 fold

19

Characterisation of 182 Fc1 (I)

KD = 36 pM

0 .0 0 0 0 0 1 0 .0 0 0 1 0 .0 1 1 1 0 0 1 0 0 0 0

0

5 0

1 0 0

1 5 0

n M

% I

nh

ibit

ion

10

0-(

X(O

D 4

50

-63

0)n

m /

Ma

x (

OD

45

0-6

30

)nm

)

1 8 2 F c 1

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

182 Fc1 EC50 178pM

• 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

Characterisation of 182 Fc1 (II)

• 182 Fc1 given as single bolus IV injection at 5,10 and 20 mg/kg

• 3 animals per time point

• Followed PK out to 7 days

• 182 Fc1 well tolerated with no adverse effects

21

Pharmacokinetics of 182 Fc1

0 5 0 1 0 0 1 5 0 2 0 0

0 .0 1

0 .1

1

1 0

1 0 0

1 0 0 0

P K p a r a 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

T im e (h )

Se

ru

m C

on

ce

ntr

ati

on

(

g/m

L)

5 m g /K g

1 0 m g /K g

2 0 m g /K g

[18

2 F

c1]

(μg

/ml)

Dose (mg/kg) Half-life (h)

5 20.9±1.3

10 19.2

20 59.9±5.3

• 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

22

CT26 Syngeneic Tumour Model

Grp 1 = PBSGrp 2 = hFc1Grp 3 = 182 Fc1Grp 4 = 10F9G2Grp 5 = rat IgG2b

DR= Day of randomization. 50 out of 70 tumours reached a mean volume of 91 ± 22 mm3

• 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

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CT26 Syngeneic Model: Results

**** p<0.0001, α=0.05, multiple comparison 2-ways ANOVA

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

Affimer Scaffold Immunogenicity Testing

Immunogenicity Assessment: Human PBMC Assay

50 healthy donors representing a broad

population mix

Collect immune cells from human blood

Test therapeutic protein e.g. Affimer

Incubate for 1 week

Analyse immune cell activation and

proliferation by flow cytometry

50 μg/ml

26

27

Human PBMC Testing Results• In silico immunogenicity of the

Affimer scaffold was determined to be low

• Affimer scaffold immunogenicity compared to Avastin (50 μg/ml)

• KLH positive control

• Positive response: SI>2 with p<0.05

• Core Affimer scaffold has a low immunogenicity potential

• Will be repeated on lead molecules

0

5

10

15

2020304050

0

10

20

30

40406080100

Positive Responses

#P

ositi v

eD

ono

rs

%P

os

iti v

eD

ono

rs

• 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 levelso 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

Summary

Acknowledgements

Avacta Life Sciences

The PD-L1 project has been supported by an Innovate UK Grant

University of Leeds (BSTG)

• Christina Rauber

• Lindsay McMorran

• Graham Spence

• Paul Shadbolt

• Rob Ford

• Andrew Wilcox

• Matt Johnson

• Emma Jenkins

• Estelle Adam

• Flo Laurent

• Marine De Jaeger

• Dino Ossola

• Ming Zhou

• Jyrki Sivula

• Emma Stanley

• Michele Writer

• Lemy Tsikna

• Anna Tang

• Mike McPherson

• Darren Tomlinson