Assessing the metabolic turnover of mAb targets in humans using Accelerator Mass Spectrometry

1. Assessing the metabolic turnover of mAb targets in humans using Accelerator Mass Spectrometry Graham Lappin Chief Scientific OfficerXceleron Inc

2. Summary • Importance of mAb-target engagement and rate of target production in the body • Is the target druggable? • Dosing regimen • Measurement of target production in the body • In the presence of mAb • Prior to mAb development • IMPACT (Innovative Measurement of Proteins using AMS to Characterise Targets) : a pre-competitive collaboration

3. Target interaction Target Downstreampharmacology

4. Target mediated disposition Receptor on target Therapeutic proteinfree inplasma Dose Kon Target is neutralised Koff Drug-receptorcomplex Kelim Elimination Jin & Krzyzanski AAPS Pharma Sci 2004: 6 (1) 1-8

5. Engagement of therapeutic antibody with target Target Plasma space mAb mAbdosed

6. Engagement of therapeutic antibody with target Target Plasma space mAb mAbdosed Elimination Target unsaturated

7. Engagement of therapeutic antibody with target Target Plasma space mAb mAbdosed In this case doseis based on concentrationof target present Elimination Target saturated

8. Rate of target production Target Plasma space Target re-synthesised mAb Depending onhow quickly the target returns then another dose of mAbrequired The rate of productionof target is the moreimportant parameter insetting dose Elimination

9. Clarification of terms Rate of target production The amount of target produced in the body and presented to the therapeuticantibody per time. Turnover rate: The amount of target removed versus the rate of target produced per time.Measurement of turnover assumes steady-state conditions apply. Steady-state conditions may not necessarily be present in every case and so to keep the terminology consistent, this presentation will use “target production”, although purists may point out the error!

10. Target burden • Understanding mAb-target engagement is fundamental for biopharmaceuticals • Target burden • Target engagement • Rate of target production • Important in ascertaining dose and its regimen • Dose required for therapeutic effect • Regimen may be unsustainable

11. Measuring rate of target production Target Plasma space mAb Measure free targetand drug-target complexover time to obtaininformation on target production calculated by inferenceusing mathematicalmodel mAbdosed

12. Measuring rate of target production • Target production rate measured in presence of mAb • Can only be measured once mAb is in the clinic • No information on intrinsic target production rate • Absence of mAb (ie “normal state”) • Absence of mAb in disease state • Target production rate may be unsuitable but only known once in the clinic • GMP manufacture of mAb required – which can be expensive

13. Measuring rate of target production • Often little to no information on target production rate prior to clinical trials. Only data are on concentration of target • Target concentration says nothing about production rate • A dose of mAb based purely on existing target concentration can be very misleading • For example, low target concentration may lead one to believe a low mAb dose will be sufficient to neutralise • If the production rate is high however, the low dose of mAb may be “swept away” by newly generated target • The amount of mAb required for some targets may be excessive • One case of a required dose of 10mg/kg/day! to neutralisethe target

14. Early target production data • Intrinsic target production rate (in absence or mAb) might indicate if target is druggable • Target production rate in healthy and disease state is also useful in assessing target druggability • Animal models useful but data in humans more reliable • Target production rate in pathological state particularly useful • In vitro will give information on mAb-target interaction but not ontarget production rate in vivo • Targets can be proteins or even cell populations. Cell-tracking studies can be very informative but also challenging to perform

15. Measuring target production prior to mAb manufacture • To measure target production rate of any biological entity (small molecule, protein or cell population) the target has to be labelled, typically isotopically • The clearance of the label is measured (using an isotopic assay) • The total amount of target is measured • Target production rate (turnover - more accurately in this case) is the product of isotopic clearance and total target concentration

16. Target production rate Plasma space Measure totalconcentrationof target

17. Target production rate Plasma space Measure presence oflabelled target over timeto calculate clearance Measure totalconcentrationof target

18. Labelling targets • “Classical” labels are high specific activity (short half-life) radioisotopes – 125I 99mTc • Short half-lives limit duration of experiments • 125I t½ = 60.25 days • 99mTc t½ = 6 hours • If administered to humans, radioactive burden can be high • For small molecules 14C is the isotope of choice • 14C t½ = 5760 years, so sensitivity is problematic for largemolecules

19. Using 14C to label proteins • 14C is a long half-life and so few time restrictions experimentally • Low energy ß-emitter • Lower radioactive burden for human subjects • 125I in particular requires “iodine wash-out” • Reduced autoradiolysis • Fewer safety issues in laboratory handling • But scintillation assay sensitivity with 14C and large molecules problematic • Requires a highly sensitive 14C assay : accelerator mass spectrometry (AMS)

20. -Decay of 14C atom Detected by scintillation counting as photons of light in photomultiplier tube Atoms separated 12C,13C and 14C atoms individually counted by differences in mass/charge and energy AMS is exquisitely sensitive to 14C Scintillation counting 0.012% of 14C decays per annum; 2.3 billion 14C atoms ≡ 1 dpm AMS 1000 14C atoms required for valid measurement Key Sample containing12C 13C and 14C atoms

21. AMS instrument 250kV AMS based at Xceleron, Germantown, Maryland

22. 14C-targets and AMS 14C 13C 13C Bioactivityassay Labelling of mAb Analysis MALDI-TOF-MS (using 13C as marker) SDS-PAGE Target administered to humans. Very low levels of radioactivity (classified asnon-radioactive study) Very low mass of target so as not to perturbnaturally occurring pool

23. Announcement of IMPACT Innovative Measurement of Proteins using AMS to Characterise Targets • A pre-competitive research collaboration between Xceleron,Hammersmith Medicines Research and the Biopharma Industry • Two targets as examples to demonstrate the technique • TNF- • T-lymphocytes • IMPACT is a proposed programme of work, supported by aconsortium of Biopharma companies

24. Therapeutic proteins for treatment of rheumatoid arthritis (RA) Target TNF-

25. Some targets in rheumatoid arthritis Abatacept T-cell Activatedmacrophage Adalimumab Etanercept Infliximab Certolizumab Golimumab Cytokines TNF- Osteoclasts Inflammation Synovial tissue Scott (2012)Clin Pharm Ther 91(1) 30-43

26. IMPACT: measurement of TNF- production Proof of principle Samples of plasma and a limitednumber of synovial fluidsamples over time Healthyvolunteers 14C-TNF- Measure total TNF- and 14C-TNF to calculatetarget production rate lipopolysaccharide (LPS)challenge RA patients

27. TNF- AMS assay SDS-PAGE Size exclusion • Assay specificity – probably better than ELISA • Assay sensitivity likely to be at least 2 orders of magnitude better than ELISA

28. Data outputs • Rate of target production in healthy volunteers and in disease state • TNF- already an established target but shows proof of concept • Nevertheless rates of production of TNF- in the absence of a therapeutic antibody have not been fully investigated • Data can be used retrospectively – ie what information would it provide on doses and dose regimens in the context of known effective therapeutic treatments

29. IMPACT: cell tracking studies Cell trackingstudies (plasmalymph) 14C 99mTc Half-life of 99mTc = 6 h Interference with the cell? Radiolytic stability? Half-life 14C = 5760 years Interference with cell less likely Likely to be radiolytically more stable Low radioactive burden in humans. Track cells in plasma and lymph (or anywherea sample can be taken) by measurement of 14C with AMS

30. T-Cell assay Blood sample 14C-labelling Cell separation Bloodlymph etc AMS

31. Data outputs • Rate of T-cell production and transfer between circulation and lymph • Comparison with 99mTc studies – are they reliable? • Longer-term tracking from systemic to lymph and perhaps back again?

32. Conclusions • Target production rate is important in assessing the druggability of the target and the dosing regimen • Usually measured via mAb-target complex and modelling, after target selection and manufacture of mAb for clinic • 14C-labelling and AMS allows target production rate to be measured prior to mAb development • Intrinsic target production rate in healthy and disease state possible • IMPACT: pre-competitive collaboration to develop methods to measure intrinsic target production rate in humans prior to full antibody development

33. Acknowledgements • Joe Balthasar (University at Buffalo) • Malcolm Boyce, Hammersmith Medicines Research, London • Members and potential members of the biopharma consortium Questions gratefully received Graham Lappin Xceleron Inc graham.lappin@xceleron.com