key technologies and challenges in pre-clinical biologics ... · phage display (cxcr2 mpls) screen...
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Key technologies and challenges in pre-clinical biologics drug discovery
Andrew BuchananEMI Training Event 4thApril 2017
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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
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mAb Platform TechnologiesStructural features of mAbs
Fab
Fc
Fv
CDR’sComplementary determining region
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Range of Biologic’s formatBispecific Antibodies
Antibody Drug ConjugatemAb Fragments
Peptide fusionsPeptide conjugates
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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
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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®)
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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
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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?
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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
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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
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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
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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?
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Status at end of Lead Isolation
✓
✓
✓
✓
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Lead Optimisation: Case Study
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Fc
Fv
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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
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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
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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