Affimer fimer Biother therapeut apeutics: ics: Th The precl - - PowerPoint PPT Presentation

Affimer fimer Biother therapeut apeutics: ics: Th The precl eclinical inical develo velopment pment and valida lidation tion of a PD PD-L1 1 anta tagonist

• nist

in mouse use

Amrik Basran Chief Scientific Officer

NGP GPT San an Diego, go, 6th

th Jun

une 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

• n immuno-oncology and immuno-

inflammation

• Research collaboration and license

deal with Moderna Therapeutics

fAb 48 kDa ScFv 24 kDa VH dAb VL dAb 12 kDa CH2 CH3 CH1 VH CL VL IgG 150 kDa

Therapeutic Protein Scaffolds

DARPins Anticalins 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 scaffolds

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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 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”

• n the tip of the virus

Loop 4 Loop 2

Affimer Gene

Loop 2 9 aa Loop 4 9 aa

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Lead Identification: Phage Selections

Acid elution

• f 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 weeks

Expression ELISA BIAcore SEC-MALLS Solubility Tm Cell assay Cross reactivity Lead Clones

Affinity Maturation

7 Formatting Immunogenicity testing Developability assessment PK & efficacy

CAR-T

Immuno-oncology Strategy

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Combination Therapies and Agonists T-cell Recruitment Drug Conjugates T-cell Tumour

Intratumoral Expression

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Pharmacokinetics

Time (h) %ID/ml Serum

0.1 1 10 100 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

• f the protein to prevent

clearance via the kidneys Affimer biotherapeutic binds to HuSA in the circulation

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Human Serum Albumin Fc Fusions

PD PD-L1 L1 Pr Prog

• gram

am

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

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Multimer Formatting: PoC With PDL1-141

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• Formatted as IgG1 Fc fusion

and expressed transiently in Expi293F cells

• Purified using PrA sepharose

followed by prep-SEC (yield ~200 mg/L)

• PDL1-251 Fc KD of ~40 pM by

Biacore

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Fc Formatting of PDL1-251

PDL1-251 Fc SEC-HPLC KD= ~40 pM PDL1-251 Fc Biacore

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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

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PD-1/PD-L1 Cell Based Assay

0.01 0.1 1 10 100 1000 10000 2 4 6

nM

Fold of induction

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

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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)

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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

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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 5 0 1 0 0 1 5 0

n M % In h ib itio n

1 0 0 -(X (OD 450-630)nm / M a x (OD 450-630)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

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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

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Pharmacokinetics of 182 Fc1

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

T im e (h )  5 m g /K g 1 0 m g /K g 2 0 m g /K g

[182 Fc1] (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

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CT26 Syngeneic Tumour Model

Grp 1 = PBS Grp 2 = hFc1 Grp 3 = 182 Fc1 Grp 4 = 10F9G2 Grp 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
• r 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

Im Immunogenicity munogenicity Tes esti ting ng

• 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

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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

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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

5 10 15 20 20 30 40 50 10 20 30 40 40 60 80 100

Positive Responses

# Positive Donors % Positive Donors

• Affimer therapeutics are an alternative to therapeutic antibodies with key

benefits:

• Generation of single digit/double digit nM binders from naïve libraries
• Easily formatted e.g. multimers and Fc fusions with high expression levels
• 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

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Summary

Acknowledgements

Avacta acta Life Sciences ences

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

Uni nive versi rsity ty of Leeds eds (BST STG) G)

• 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