Bioassay development for complex molecules- Challenges with Bispecific

Molecules

Bhavin Parekh, Ph.D.Group Leader-Bioassay Development

Eli Lilly and CompanyIndianapolis, IN 46221

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Outline

Background Bioassay expectations

Developing a MOA-based strategy for bispecific molecules

Case Study: Challenges in development, control and analysis of bioassays for a bispecific antibody

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Regulatory expectations for Bioassays

Reflective of the mechanism(s) of action (MoAs) of the product

Sensitive to structural changes that can impact product safety and efficacy

Stability indicating (detection of degradation)Well controlled (low CV, tight precision, etc.)Usable as a QC release assay under GMP quality

systemBinding based assay may be acceptable to support

early clinical phase. Need MOA-reflective, functional assay by pivotal.

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Bispecific antibodies come in many flavors

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Different targets of Bispecific antibody

Both targets are soluble ligands

One target is soluble ligand and other is

cell surface receptor

Both targets arecell surface

receptors, buton different cells

Both targets arecell surface receptors,

buton the same cell

Influences the strategy of what type/number of potency assay are relevant

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Potency Assay strategy for BsAbs

BsAbs have the unique potential to mediate two types of biological effects (Additive or synergistic) Additive effects

Biological outcome with the BsAb is the sum of the two independent target When the targets bind to two cell types, independent two bioassays seem

appropriate When the targets bind to the same cell type, a single bioassay is appropriate

mAb A (80%)mAb B (70%)

BsAb (50%)

% v

iabl

e ce

lls

100%

Conc.

BsAb = mAb A + mAb B

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Potency Assay strategy for BsAbs

BsAbs have the potential to mediate two types of biological effects (Additive or synergistic)Synergistic effectsBiological effect of BsAb is greater than the sum of the two

independent targets.Can be manifested at different biological levels (physiological

and/or cellular)Physiological synergy

Targets can be different cell types (e.g., B-cell and T-cells) with different signaling pathways BUT provide a more synergistic physiological outcome revealed in a preclinical animal model

This synergistic outcome cannot be reflected in a unique combined assay. Thus, two separate and distinct bioassays is appropriate

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Potency Assay strategy for BsAbs

Synergistic effectsCellular synergy

Targets present on same cell populationSpecific synergy assay is needed

mAb A (80%)

mAb B (70%)

BsAb (10%)

% v

iabl

e ce

lls

100%

BsAb > mAb A + mAb B

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Bioassay strategy for Bispecific antibody: How many assays?

Anti-receptor 2

Anti-receptor 1VL2 VL2

VH2VH2

P P

P P

Synergistic assayAntibody B (Bispecific)

Receptor 2 assay

Receptor 1 assay

Bioassay Strategy:

- If Receptor 1 and Receptor 2 have similar signaling pathway: Synergistic assay

- If different signaling pathway: Two individual Cell based assay

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Case Study: BsAb

Targets 2 receptors (M and E) involved in cancerCan be expressed on same cells

Receptors have been targeted independently with specific mAbs

In cells expressing both receptors, signaling pathways via receptor M and E can cross talk

Receptor have been shown to hetero-dimerize

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Case Study: Bispecific Ab binding to two receptor targets (M and E) on the same cell

Objectives:

Develop potency assay strategy for BsAb that is reflective of the synergistic MOA

mAb for Receptor E (Ab 2)Combo, mAb M+E

BsAb

mAb for Receptor M (Ab 1)

ControlReceptor M

Receptor E

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Requirements for a synergy assay for BsAB

Cell line has to express both receptors Readout has to be sensitive to inhibition of both receptors BsAb activity greater than

Individual mAb M and mAB E Combinaton of mAb M and mAb E

mAb M + mAb E

mAb MmAb E

BsAb% v

iabi

lity

100%

Conc.

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0.00

2000000.00

4000000.00

6000000.00

8000000.00

10000000.00

12000000.00

0.01 0.10 1.00 10.00 100.00

RLU

Concentration (nM)

Synergy assay- Finding the correct cell line

Receptor E AbControl

Receptor M Ab

Combi. TherapyRecep.M Ab +

Recep.E Ab

Bispecific Antibody

Cell line G provides response to Receptor M and not Receptor E, thus can not provide mechanistically relevant synergistic assay.

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0.00

1000000.00

2000000.00

3000000.00

4000000.00

5000000.00

6000000.00

0.01 0.10 1.00 10.00 100.00

RLU

Concentration (nM)

Receptor M AbControl

Receptor E Ab

Combi. TherapyRecep.M Ab +

Recep.E AbBispecific Antibody

Cell line F provides response to Receptor E and not Receptor M, thus can not provide mechanistically relevant synergistic assay.

Synergy assay- Finding the correct cell line

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In cell line F, BsAb shows superior effect from individual Abs and from Combo.

Synergy assay- Finding the correct cell line

Lum

ines

cenc

e Receptor M Ab

Combo (M + E Abs)BsAb

Receptor E Ab

Antibody conc. (nM)

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Potency analysis of typical mAbs vs. BsAb

a

cb

d

a

d

b

Concentration

Sign

al

y = d +a - d

1 + (conc/c)b a- lower asymptoted- upper asymptoteb- slopec- EC50

mAb M

mAb E

BsAb

% v

iabi

lity

100%

Conc.Synergistic assay profile Change in EC50, asymptotes Not amenable to parallelism test How do we adequately capture the biological change? How would dose-response curve shapes change upon loss of

stability at either sides

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Analysis of non-parallel curves

Well documented in the field of pharmacologyStudy of partial agonist or antagonistBiological activity is represented by change in EC50 and

relative change in asymptote (referred to as Efficacy)

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New approach for determining relative potency/efficacyfor BsAb with synergistic activity

- BsAb changes two different aspects of the dose response (EC50 and asymptote ratio)

- For Bispecifics with different asymptotes: Efficacy = Proliferation reduction/ EC50

= (Upper asymptote/Lower asymptote)---------------------------------------------------

EC50

Cell line Ligand AnalyteA (Upper asympt.)

D (Lower asympt.)

Proliferation reduction (PR=A/D)

EC50 (nM)

Efficacy factor (EF=PR/C)

FNo

ligand Recep. E Ab 6370000.00 2200000.00 2.89 0.56 5.16

Recep. M Ab 6180000.00 6100000.00 1.01 N/A N/A

Combo-1 6700000.00 1010000.00 6.63 0.72 9.21

BsAb B 6290000.00 702000.00 8.96 0.16 56.00

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AcknowledgementsBioassay groupPiyush VyasMaroun BeyrouthyDarren KamikuraSara LallyDenise LyonsJane SternerSharon SibleyJeanne HelmreichClaudius TapiaJason LehmanTeresa LedfordGretchen Smith

StatisticsKristi GriffithsEric AdamecShu ZhengKevin Guo