xceleron - all rights reserved ©2012 graham lappin chief scientific officer xceleron inc

Xceleron - all rights reserved ©2012

Assessing the metabolic turnover of mAbtargets in humans using Accelerator Mass

Spectrometry

Graham LappinChief Scientific Officer

Xceleron Inc


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


Target interaction

Target Downstreampharmacology


Target mediated disposition

Jin & Krzyzanski AAPS Pharma Sci 2004: 6 (1) 1-8

Drug-receptorcomplex

Receptor on target

Kon Koff

Elimination

Kelim

Therapeutic proteinfree inplasma

Dose Target is neutralised


Target

Engagement of therapeutic antibody with target

mAb

Plasma space

mAbdosed


Target

mAb

Plasma space

mAbdosed

EliminationTarget unsaturated



Target

mAb

Plasma space

mAbdosed

Target saturated Elimination

In this case doseis based on concentrationof target present



Target

Rate of target production

mAb

Plasma space

Elimination

Depending onhow quickly the target returnsthen another dose of mAbrequired

Target re-synthesised

The rate of productionof target is the moreimportant parameter insetting dose


Clarification of terms

Rate of target productionThe 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 soto keep the terminology consistent, this presentation will use “target production”, although purists may point out the error!


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


Target

mAb

Plasma space

mAbdosed

Measure free targetand drug-target complexover time to obtaininformation on target production calculated by inferenceusing mathematicalmodel

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


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



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 on

target production rate in vivo Targets can be proteins or even cell populations. Cell-tracking

studies can be very informative but also challenging to perform


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


Target production rate

Plasma spaceMeasure totalconcentrationof target


Plasma spaceMeasure presence oflabelled target over timeto calculate clearance

Measure totalconcentrationof target

Target production rate


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


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)


AMS is exquisitely sensitive to 14C

AMS

Atoms separated 12C,13C and 14C atomsindividually countedby differences in mass/charge

and energy

-Decay of 14C atomDetected by scintillation counting as photons

of light in photomultiplier tube

0.012% of 14C decays per annum; 2.3 billion 14C atoms ≡ 1 dpm

Key

Sample containing 12C 13C and 14C atoms

1000 14C atoms required for valid measurement

Scintillation counting


AMS instrument

250kV AMS based at Xceleron, Germantown, Maryland


14C-targets and AMS

Labelling of mAb

14C13C

13C

Bioactivityassay

AnalysisMALDI-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


Announcement of IMPACT

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

Innovative Measurement of Proteins using AMS to Characterise Targets


Therapeutic proteins for treatment of rheumatoid arthritis (RA)

Agent Type

Adalimumab Recombinant human IgG1 mAb

Etanercept Soluble TNF-receptor fusion protein

Infliximab Chimeric IgG1

Certolizumab Recombinant humanised Fab’ fragment of anti-TNF-antibody coupled to PEG

Golimumab Recombinant human IgG1 mAb

Target TNF-


Some targets in rheumatoid arthritis

T-cell Activatedmacrophage

CytokinesTNF-

Osteoclasts

Synovial tissue

AdalimumabEtanerceptInfliximabCertolizumabGolimumab

Abatacept

Scott (2012)Clin Pharm Ther 91(1) 30-43

Inflammation


IMPACT: measurement of TNF- productionProof of principle

Healthyvolunteers

14C-TNF-

lipopolysaccharide (LPS)challenge

RA patients

Samples of plasma and a limitednumber of synovial fluidsamples over time

Measure total TNF- and14C-TNF to calculatetarget production rate


TNF- AMS assay

Size exclusion

SDS-PAGE

Assay specificity – probably better than ELISA Assay sensitivity likely to be at least 2 orders of

magnitude better than ELISA


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

Data outputs


IMPACT: cell tracking studies

99mTcCell trackingstudies (plasmalymph)

Half-life of 99mTc = 6 hInterference with the cell?Radiolytic stability?

14C

Half-life 14C = 5760 yearsInterference with cell less likelyLikely 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


T-Cell assay

Blood sample

Cell separation

14C-labelling

AMS

Bloodlymph etc


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?

Data outputs


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


Joe Balthasar (University at Buffalo) Malcolm Boyce, Hammersmith Medicines Research, London Members and potential members of the biopharma consortium

Acknowledgements

Questions gratefully received

Graham LappinXceleron [email protected]

xceleron - all rights reserved ©2012 graham lappin chief scientific officer xceleron inc

Documents

target druggable

target re

generated target

mabtarget engagement

measuring target production

mabtarget interaction

plasma dose target

low target concentration