affimer fimer biother therapeut apeutics ics th the precl
play

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

Aug 04, 2023 •308 likes •616 views

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 onist in mouse use th Jun NGP GPT San an Diego, go, 6 th une 2017 Amrik

  1. 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 onist in mouse use th Jun NGP GPT San an Diego, go, 6 th une 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 immuno- inflammation • Research collaboration and license deal with Moderna Therapeutics 2

  3. Therapeutic Protein Scaffolds V H C H 1 • Most successful class of protein therapeutics V L C L ScFv fAb • But IgGs are large and limited routes of C H 2 24 kDa 48 kDa administration C H 3 • Difficult manufacturing/disulphides/fragment stability IgG V H dAb V L dAb 150 kDa 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 administration (e.g. topical) Non- IgG based scaffolds 3

  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 • 4

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

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

  7. 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

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

  9. Pharmacokinetics 100 Therapeutic window %ID/ml Serum 10 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

  10. Serum Half-life Extension Technologies -S- Human Serum PEGylation Fc Fusions 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

  11. PD PD-L1 L1 Pr Prog ogram am

  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, 2013 12

  13. 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 • Affimer binders compete for human PD-1/CD80 epitopes on PD-L1 13

  14. Multimer Formatting: PoC With PDL1-141 14

  15. 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 K D of ~40 pM by Biacore K D = ~40 pM 15 15

  16. 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 μ M • PDL1-251 Fc has an EC 50 ~40-50 6 nM (~25 fold improvement with Fold of induction formatting) 4 mAb 29E.2A3 • Lead Affimers binders are now PDL1-251 Fc undergoing affinity maturation, 2 PDL1-251 linker optimisation etc 0 0.01 0.1 1 10 100 1000 10000 16 nM

  17. Mouse PD-L1 Program mPD-L1 Biacore • Human PD-L1 Affimer App K D = 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 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 182 Fc1 SEC-HPLC chromatography followed by preparative SEC • Final purified yield >100mg/L yield, purity >95% (SEC-HPLC) > 95% purity 18

  19. 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 1 5 0 1 0 0 -(X (OD 450-630)nm / M a x (OD 450-630)nm ) A n ti m u P D -L 1 (1 0 F 9 .G 2 ) ~10 fold 1 8 2 F c 1 % In h ib itio n 1 0 0 • Competition against PD-1 182 Fc1 EC 50 178pM increased ~100 fold 5 0 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 19 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. Pharmacokinetics of 182 Fc1 1 0 0 0 5 m g /K g • 182 Fc1 given as single  [182 Fc1] ( μ g/ml) 1 0 m g /K g 1 0 0 bolus IV injection at 5,10 2 0 m g /K g 1 0 and 20 mg/kg 1 • 3 animals per time point 0 .1 • Followed PK out to 7 days 0 .0 1 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 20 59.9±5.3 21

  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. Im Immunogenicity munogenicity Tes esti ting ng

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

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

  27. Human PBMC Testing Results • In silico immunogenicity of the Affimer scaffold was determined Positive Responses to be low 50 100 40 80 • Affimer scaffold immunogenicity 30 60 % Positive Donors # Positive Donors compared to Avastin (50 μ g/ml) 20 40 20 40 • KLH positive control 15 30 • Positive response: SI>2 with 10 20 p<0.05 5 10 • Core Affimer scaffold has a low 0 0 immunogenicity potential • 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

More recommend