Key technologies and challenges in pre-clinical biologics drug discovery

Andrew BuchananEMI Training Event 4thApril 2017

From Discover to DevelopmentA simple paradigm…

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• First select the right candidate molecule!

Isolate

candidate

drug

Manufacture

candidate

drug

Find

patient

Put product in

patientNew Medicine

mAb Platform TechnologiesStructural features of mAbs

Fab

Fc

Fv

CDR’sComplementary determining region

Range of Biologic’s formatBispecific Antibodies

Antibody Drug ConjugatemAb Fragments

Peptide fusionsPeptide conjugates

mAb Platform TechnologiesLarge libraries of human antibodies

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non-immunised

humans, 7-70 yrs

Isolated B cells

VH and VL genes

assembledCloning

VH and VL genes

amplified PCR

Vaughan et al (1996) Nature BioTechnology

Lloyd et al, 2008 PEDS

mAb Platform TechnologiesIn vitro large libraries of human antibodies

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Display Technology for mAbs

• Very high affinity requirement (<10 pM)

• Antigen highly conserved between species

• Toxic or poorly immunogenic

• Over 40 clinical and preclinical therapeutic

antibodies, including adalimumab (Humira®) and

belimumab (Benlysta®)

mAb Platform TechnologiesIn vivo animal immunisation

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Immunisation Extract B cells from

lymph nodes & spleen

Fuse with myeloma cells

Screen and select

hybridomasHybridoma for mAbs

• First in class – early lead

• Complex cell surface targets (GPCR’s, ion channels)

• Over 13 approved therapeutic mAbs

Candidate Drug Target Profile

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Target What do I want to bind?

Specificity Do I want it to bind anything else?

Format What will the antibody look like?

Species cross reactivity What species do I need to cross react with?

Potency What effect do I want my antibody to have and how will I measure it?

Affinity How will I know when it is good enough?

Developability Does it have good biophysical stability, expression and PK characteristics?

Lead Isolation: Case Study

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Case Study: CXCR2

Target

Class A GPCR

Activated by the binding of a number of CXC chemokines with the conserved ELR

sequence motif

Goal: Generate CXCR2 antagonistic antibodies

Challenges for antibody discovery: Small extracellular regions, poor expression

and unstable in purified form

Rossant MAbs. 2014;6(6):1425-38

Approaches for Isolation of CXCR2 antibodies

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Immunisation

– Immunisation of mice with HEK cells over-expressing human

CXCR2 (> 1x105/cell)

Phage Display

– Magnetic proteoliposomes (MPLs) ensure CXCR2 is:

• In the correct orientation

• Pure

• Highly concentrated

MagneticBead

CXCR2

LipidMembrane

Antibody

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Anti-CXCR2 mAb Functional Assay Cascade

Phage Display(CXCR2 MPLs)

Screen CXCR2 v CXCR3 binding(FMAT)

Primary screen for functionIL-18 competition binding assay (FMAT)

Leads

Secondary screen for functionInhibition of IL-18 or Gro-(TANGO arrestin assay)

Specific Antibodies

Potent, species cross reactive antibodies

Mouse Immunization(HEK-CXCR2 cells)

1373

93

5

70

9,800

303

1

3

1,536

1% binding mAbswere functional

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Anti-CXCR2 mAb Functional Assay Cascade

Phage Display(CXCR2 MPLs)

Screen CXCR2 v CXCR3 binding(FMAT)

Primary screen for functionIL-18 competition binding assay (FMAT)

Leads

Secondary screen for functionInhibition of IL-18 or Gro-(TANGO arrestin assay)

Specific Antibodies

Potent, species cross reactive antibodies

Mouse Immunization(HEK-CXCR2 cells)

1373

93

5

70

9,800

303

1

3

1,536mAb IL-18

IC50 (nM)

Gro-α

IC50 (nM)

X-1 2.2 3.8

X-2 1.6 6.2

X-3 0.5 1.1

X-4 0.08 0.07

HY 0.2 0.4

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How can antibodies inhibit?Determining mechanism of action

Competitive Allosteric

Antibodies with distinct modes of action identified from different lead isolation approaches

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Hybridoma derived antibody

Antagonist

Phage display derived antibody

Allosteric modulator

X-3HY

Target What do I want to bind?

Specificity Do I want it to bind anything else?

Format What will the antibody look like?

Species cross reactivity What species do I need to cross react with?

Potency What effect do I want my antibody to have and how will I measure it?

Affinity How will I know when it is good enough?

Developability Does it have good biophysical stability, expression and PK characteristics?

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Status at end of Lead Isolation

✓

✓

✓

✓

Lead Optimisation: Case Study

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Fc

Fv

Why do we want high affinity antibody drug candidates?

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• To meet the needs of patients

and physicians

– High affinity to engage target

over prolonged periods

– Minimise dose required

• Target 100 pM affinity

Minter et al. 2013 BJP 168:200

Anti-GM-CSF Receptor for Rheumatoid Arthritis

See Figure 1 in

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Affinity Maturation Platforms

Parsimonious Mutagenesis

• Single-point mutations for screening

• Combine all improved AA changes

Block Mutagenesis:

Library based approach

Target multiple CDR positions

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Screening for improved affinity and potency

See phase II progress in Burmester et al. 2017 Ann

Rhem Dis 0:1-11

GM-CSF R

mAb

Affinity Shape change IC50

Parent 71 nM -

547D04 29 pM 41 pM

VH VL

DevelopabilityCritical to ensure the desired formulation, PK and exposure

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Dobson et al. Sci Reports 2016 6:38644

SEC-HPLC

Single dose rat PK

mAb IHC on normal human tissue

Candidate Drug Target Profile

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Summary

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• Key features of mAbs and the range of formats

• The use of a Candidate Drug Target Profile as a guide

• Case studies of lead isolation and optimisation:

– Power of combining antibody platform and functional screening

• Importance of ‘developability’ for clinical development

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Scenario’s: What would you do?

Ligand or Receptor

Species of interest

Human specific

KD 100 nM

mAb aggregates

ADA response

Can’t reproduce published data

Short T1/2