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

Transcription

1 Affimer Biotherapeutics: The preclinical development and validation of a PD-L1 antagonist in mouse NGPT San Diego, 6 th June 2017 Amrik Basran Chief Scientific Officer

2 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 immunoinflammation Research collaboration and license deal with Moderna Therapeutics 2

3 Therapeutic Protein Scaffolds C H 1 V H 3 C H 2 C H 3 IgG 150 kda Anticalins C L V L fab 48 kda IgG based scaffolds DARPins V H dab Non- IgG based scaffolds 12 kda ScFv 24 kda V L dab 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)

4 4 Affimer Technology Based on Stefin A, a human intracellular protein 1/10 th 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 (1x10 10 ) Utilise phage display to identify binders

5 Library Generation: Phage Display Affimer Gene Loop 2 9 aa Loop 4 9 aa Loop 4 Loop 2 Protein displayed on the tip of the virus Affimer library containing over 10 billion different gene sequences is then packaged with viral DNA Microbial host (E. coli) 5 DNA encoding the Affimer gene and the virus. Affimer gene and protein now linked

6 Lead Identification: Phage Selections Selection Pressure Binding Step Wash Step Target Antigen DNA Repeat Acid elution of the phage +Antigen -Antigen Infect and amplify in E. coli

7 The Process: Lead Characterisation Assay Development Antigen biotinylation and QC Phage Screening (cross reactivity) Sub-clone binders ~5-7 weeks Screening: BIAcore ELISA etc DNA Sequencing Expression ELISA BIAcore SEC-MALLS Solubility Tm Cell assay Cross reactivity Affinity Maturation Lead Clones 7 Formatting Immunogenicity testing Developability assessment PK & efficacy

8 Immuno-oncology Strategy Combination Therapies and Agonists T-cell Recruitment T-cell CAR-T Tumour Drug Conjugates 8 Intratumoral Expression

9 %ID/ml Serum Pharmacokinetics 100 Therapeutic window Short serum half-life ~0.5hrs, due to renal clearance (~<60kDa) - acute indications - in vivo imaging reagents Time (h)

10 Serum Half-life Extension Technologies -S- Fc Fusions Utilising IgG-FcRn recycling to maintain high serum levels PEGylation Increased hydrodynamic size of the protein to prevent clearance via the kidneys Human Serum Albumin Affimer biotherapeutic binds to HuSA in the circulation 10

11 PD-L1 Program

12 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,

13 Anti-PD-L1 Binders: Production in E. coli 13 Identified a range of unique sequences Ni-NTA purified (>95%) and expression levels ~ mg/l at 15 ml scale Affimer binders compete for human PD-1/CD80 epitopes on PD-L1

14 14 Multimer Formatting: PoC With PDL1-141

15 Fc Formatting of PDL1-251 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 K D of ~40 pm by Biacore PDL1-251 Fc PDL1-251 Fc SEC-HPLC PDL1-251 PDL1-251 Fc Biacore K D = ~40 pm 15 15

16 Fold of induction PD-1/PD-L1 Cell Based Assay Engineered Jurkat cell based signalling assay involving binding between two cells (Promega) PDL1-251 monomer has an EC 50 ~1.1 μm 16 PDL1-251 Fc has an EC 50 ~40-50 nm (~25 fold improvement with formatting) Lead Affimers binders are now undergoing affinity maturation, linker optimisation etc nm mab 29E.2A3 PDL1-251 Fc PDL1-251

17 Mouse PD-L1 Program Human PD-L1 Affimer antagonists do not bind mouse antigen mpd-l1 Biacore App K D = 316 pm 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 mpd-l1 Competition ELISA IC 50 = 20 nm 17

18 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 chromatography followed by preparative SEC Final purified yield >100mg/L yield, purity >95% (SEC-HPLC) > 95% purity 182 Fc1 SEC-HPLC 18

19 % In h ib itio n (X (O D )n m / M a x (O D )n m ) Characterisation of 182 Fc1 (I) Formatting of the Affimer protein significantly increase binding affinity K D = 36 pm Improvements most likely due to avidity effects Biacore binding improved ~10 fold A n ti m u P D -L 1 (1 0 F 9.G 2 ) F c 1 Competition against PD-1 increased ~100 fold Fc1 EC pM n M

20 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

21 [182 Fc1] (μg/ml) S e r u m C o n c e n tr a tio n ( g /m L ) Pharmacokinetics of 182 Fc1 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 F c 1 in m ic e Fc1 given as single bolus IV injection at 5,10 and 20 mg/kg 3 animals per time point m g /K g 1 0 m g / K g 2 0 m g / K g Followed PK out to 7 days 182 Fc1 well tolerated with no adverse effects T im e (h ) Dose (mg/kg) Half-life (h) ± ±5.3

22 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 mm 3

23 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

24 Immunogenicity Testing

25 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: 25 In silico (identify T-cell epitopes) In vitro T-cell assays (e.g. human PBMCs, DC:T-cells) Humanised mice models

26 Immunogenicity Assessment: Human PBMC Assay Test therapeutic protein e.g. Affimer Collect immune cells from human blood 50 μg/ml 50 healthy donors representing a broad population mix Incubate for 1 week Analyse immune cell activation and proliferation by flow cytometry 26

27 # Positive Donors % Positive Donors 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) Positive Responses KLH positive control Positive response: SI>2 with p< Core Affimer scaffold has a low immunogenicity potential 0 0 Will be repeated on lead molecules 27

28 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

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

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