Antibody-like biologics screening, design, formatting

and profilingErik Depla, PhD

Director Biology, Orionis Biosciences NV

VIB training event ‘Drug Discovery’ 1 March 2018

Biologics: history

• Vaccines were the first biologics• 1796: Jenner took pus from the hand of a milkmaid with cowpox, scratched it into the arm of

an 8-year-old boy,…• practice of “vaccination” for small pox reported to go back to 10th century Chinese medicine

• Insulin the next major breakthrough• Animal insulin commercialized early 1920s• Human, E. coli derived, insulin commercialized early 1980s

• Antibodies• 1950s: polyclonal IVIG• 1975: hybridoma technology (Köhler and Milstein)• 1986: first approval: OKT3 murine IgG2a CD3 specific transplant rejection drug• 1998: first anti-TNF approval: infliximab

Evolving biologics: anti-TNF

Biologics: a large market• global biologics market size: USD 276.6 billion in 2015• anticipated to reach USD 399.5 billion by 2025

Brand name Sales 2015 (b$) company biologic indication

Humira 14.01 Abbvie anti-TNF immune

Rituxan 7.33 Roche anti-CD20 oncology

Lantus 7.09 Sanofi insulin diabetes

Avastin 6.95 Roche anti-VEGF oncology

Herceptin 6.80 Roche anti-HER2 oncology

Remicade 6.56 Janssen anti-TNF immune

Prevnar 6.21 Pfizer pneumococcal infectious

Enbrel 5.36 Amgen anti-TNF immune

Neulasta 4.72 Amgen GCSF oncology

Novorapid 3.98 Novo Nordisk insulin diabetes

Biologics: a manufacturing, formulation anddistribution challenge• Top selling drug Humira: a lot of assumptions but impressing numbers

• 14 billion dollar sales in 2015• ≈3100 dollar/dose 4.5 million prefilled syringes/pens • standard maintenance dose: 40 mg

• samples or QC• retain samples for documentation• discounts on prices• expired product• overfill of syringes… > 200 kg of pure protein

• 100 mg/ml solubility >2000 L drug substance• Assuming 1 g/L production >200,000 L of cell culture• Stable at 2-4°C in solution 18 months

The perspective of the manufacturer, regulator and society• produce, store at acceptable cost?• new could carry unknown risks!• safety and immunogenicity?

Drug discovery: think early also aboutcommercial useThe science/disease perspective• what should it do to result in

treatment?• new could be better!

Antibody and antibody like scaffolds

Antibody-like biologics – platform selection

• Antibody, antibody domain or scaffold• Any format may do for target validation• Drug discovery: antibodies and antibody domains/fragments dominate the

market and pharma industry/regulatory experience• Newer formats or scaffolds: intellectual property may be limiting

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antibody domain/fragment

Fc-mediated effector functions X

bispecific/multispecific X

monovalency required X

bivalency X X

multivalency X

chemical modification especially DAR of 1 X

Antibody-like biologics – immunization?

2 Immunization (mouse human)• historical immunity (infection)• auto-immune• active

B-cells• spleen• lymph nodes• blood/PBMC

Antibody-like biologics – immunization?

2

B-cells• spleen• lymph node• blood/PBMC

PCR library• phage, virus,• yeast, bacteria, …

Immortalization• hybridoma fusion

FACS sorting B-cells +labelled antigen

INVOLVES SELECTION

Antibody-like biologics – immunization?

2

Immortalization• hybridoma fusion• EBV

FACS sorting B-cells +Labelled antigen

PCR library• Phage, virus,• yeast, bacteria, …

Very high diversitySelect with antigen enrich for specificityOpportunity and risk

Learn andadapt

Screening andcharacterisation• binding• epitope• Competition• ligand• benchmark

• function• …

Antibody-like biologics – immunization?

• Non-immune libraries:• “Naive” library: pool of antibody (VH, VL, VHH or Fab) repertoire from a few individuals• Synthetic libraries: randomized CDRs in a standard framework (e.g. human IgG-Fab, VHH)

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immunization “naive” library synthetic library

immunization time 1-3 months none none

affinity maturation in vivo more likely needed to reach desired affinity

epitope diversity may be limited(dominance, homology)

less limited only limited by size of library

humanization required in most cases not if library based on human(ized) framework

sequence liabilities likely both in framework and CDRs not in framework, CDR design important

Synthetic library: an example

• McMahon et al., 2018 (Nature Structure & Molecular Biology)• platform, library and associated protocols freely available for non-profit research

Antibody-based biologics – strategy

• Draft a profile of your ideal (domain/fragment) antibody• Mode of action

• antagonistic, agonistic, binding (e.g. if to be used for targeting of warheads), …

• Desired cross-reactivity• non-human primate orthologue: preclinical safety testing• mouse/rat orthologue: animal models (if not surrogate antibody may be needed)• family members/paralogues: desired or not

• Epitope and affinity (probably related to mode of action)• Competition assays

• In vitro potency• In which assay and is this assay biologically relevant

• …

3

Antibody-based biologics – strategy

• Design your antibody generation and screening cascade• Antibody screening: the needle in the hay stack or worse

• It all starts with antigen quality and quantity

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Antibody-based biologics – strategy

• sufficient antigen (density) of high quality needed (immunization and/or screening)• Protein: often commercially available

• ideal for soluble antigens or (large) extracellular domains• QC for purity and conformation (e.g. ligand or receptor binding)• avoid immunogenic fusion domains or tags or design counter-selection/screening strategy

• Cells typically present native antigen• fit for (multi)membrane proteins but are “dirty”

• syngeneic recombinant cells may overcome the problem• select high expression pools or clones with proven functional expression• multiple backgrounds required for screening (e.g. HEK and CHO)• VLPs may be alternative

• peptides for linear epitopes (conjugation needed for immunogenicity)

• DNA for immunization• ideally fit for multi-membrane spanning proteins but also easy for soluble antigens• QC for functional expression (e.g. ligand binding) and preferably high expression• optionally combine with cell or VLP boost

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Antibody-based biologics – RSV example• Respiratory syncytial virus

• only replicates in the airways• enveloped virus: F, G and SH protein

• F and G validated targets for neutralization• F protein: pre- and post-fusion conformation

• vast majority of mAbs does not neutralize• 2 major serotypes: A and B

• Ablynx’ ALX-0171• Drug for treatment: high dose to be delivered fast to the lung

• nebulization high solubility and stability VHH domain• Potency ≥ palivizumab (i.m. antibody for prophylaxis) both on A and B serotype

• most conserved F-protein preferred target • antigen sources typically serotype A

• F protein: pure but in post-fusion state• Viral stock: mix of pre- and post-fusion but limited purity (other viral/host cell proteins)

Antibody-based biologics – RSV example

Post-fusion proteinSerotype A

Inactivated virusSerotype Aimmunization

Selection/screening(binding)

Post-fusion proteinSerotype A

Viral stockSerotype A

Cross-over increase chance of finding the right antibody: epitope and/or conformational relevance

Viral stockSerotype A

Sequence analysis Remove redundant clones, analyze sequence diversity and reduce complexity

Analyze your sequences

After Kendrick et al., Biotechnol Rep 2015

v

• Alignment family/lineage analyses

• Family members do recognize the same epitope• range of affinities

• in general largest difference in off-rate: measure without purifying (e.g. with SPR or BLI)• correlated with potency if competing with (large) protein type of ligand

• pick one family member (best off-rate) for functional screen

Antibody-based biologics – RSV example

Post-fusion proteinSerotype A

Inactivated virusSerotype Aimmunization

Selection/screening(binding)

Post-fusion proteinSerotype A

Viral stockSerotype A

Screening: function Viral neutralizationassay serotype A & B

Cross-over increase chance of finding the right antibody: epitope and/or conformational relevance

medium-throughput purification using tag reduce bacterial contaminant interference

Viral stockSerotype A

testing properties SolubilityNebulization stress

Sequence analysis Remove redundant clones, analyze sequence diversity and reduce complexity

Antibody-based biologics – RSV example

• Monovalent VHH does not meet potency requirements

Serotype A neutralization of prototype virus Serotype B neutralization of prototype virus

After Detalle et al., AAC 2015

Antibody-based biologics – RSV example• Trivalent VHH is ideally fit to neutralize a multivalent target on a virus

Serotype A neutralization of prototype virus Serotype B neutralization of prototype virus

After Detalle et al., AAC 2015

Nb017 ALX-0171

Antibody-based biologics – RSV example

• ALX-0171: highly potent antiviral for various RSV clinical isolates

• Solubility, stabilitity, production yield,… currently in clinical testing as nebulized treatment administered to infants with severe RSV infection

After Detalle et al., AAC 2015

Antibody-based biologics – ion channel case• Kv1.3 (immunology target: critical for T-cell activation)

• Multimembrane spanning protein forming homotetramer• 3 small extracellular domains• recombinant protein: not available• (very) close family members• relevant animal model: rat

• Key attributates desired profile• Cross-reactive to rat Kv1.3 and non-human primate• Potency as good as known sea anemone toxin benchmark

• better selectivity against neuronal and cardiac family members

Antibody-based biologics – ion channel caseKv1.3 DNA

Human Kv1.3

huKv1.3 - HEK293

immunization

Screening: binding

Counterscreene.g. against Kv1.5

Screening: function

Selection

Functional assayelectrophysiology

DNA prime

Rec Caki cell boosthuKv1.3

huKv1.3 - CHO

CHO: parent versus recombinant

HEK293: parentversus recombinant

Limit to single boost!

huKv1.3 - HEK293

huKv1.3 - CHO

Adapted from WO2015193452

Alternative for cell = VLP

Functional assayT-cell

Higher throughput

Lower throughput

Sequence analysis Remove redundant clones, analyze sequence diversity and reduce complexity

Antibody-based biologics – ion channel case• Very few hits only

• good human/cyno cross-reactivity• low rat cross-reactivity

• go back to immunizations and/or screenings and introduce rat/human cross-selections• or start formatting: bivalent construct has improved rat cross-reactivity (avidity effect)

Ablynx; 8th Annual Proteins & Antibodies Congress 2015

Antibody-based biologics – ion channel case• Increase potency formatting

Ablynx; 8th Annual Proteins & Antibodies Congress 2015

Antibody-based biologics – ion channel case• Key attributates desired profile

• Cross-reactive to rat Kv1.3 and non-human primate• Potency as good as known toxin benchmark• Strong selectivity against neuronal and cardiac family members

• + in vivo data

Ablynx; 8th Annual Proteins & Antibodies Congress 2015

Ready for (pre)clinical development?

Expect questions on:• Manufacturability?• PK?• Immunogenicity? (need for humanizationmay affect potency,

production yield, product stability,…)

Analyze sequences

After Kendrick et al., Biotechnol Rep 2015

v

• Alignment family/lineage analyses

• Family members do recognize the same epitope• range of affinities

• in general largest difference in off-rate: measure without purifying (e.g. with SPR or BLI)• correlated with potency if competing with (large) protein type of ligand

• range of production yields easy win?

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• Post translational modification risks : avoid or knock-out if needed• N-glycosylation (NXS, NXT)• Free cysteine• Pyroglutamate (N-terminal E or Q)• Methionine oxidation• Iso-Asp formation (DX or NX with X = G > S/T)• …

Analyze sequences: liabilities6

• Proteins interact• Dimerisationmultimerisation soluble aggregates precipitation

• OD320 should be close to “0” if not larger soluble multimers/aggregates may be present• analytical size exclusion chromatography: more detailed view

• Freeze/thaw stability• Mind the PBS pH drop

• Solubility• Formulation

• 0.05% Tween-80• citrate buffers• histidine buffers

Analyze the protein: liabilities7

Antibody-based biologics – protein half-life

• Protein half-life• Renal clearance: filtration cut-off about 60 kD• Catabolism by cells

• aspecific uptake• specific but target independent uptake (e.g. mannose receptor)• target dependent uptake

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Based on Sthrohl et al., Biodrugs 2015

HSA

transferrin

IgG1,2,4

IgG3IgA monomer

RBP

Factor H

Fa ctor VII

IgEFa ctor IX

Fibrinogen

IFNa

CRP

IgM

IL-2Thyroglobulin

G-CSF

Factor VIIA

PYY3-860.13

IGF-1hGH

GLP-1

0

100

200

300

400

500

600

1 3 9 27 81 243 729 2187

hour

s

MW

serum half-life of proteins

Protein recycling2-3 weeks

Size > renal filtrationcut-off

0.5- 1 week

Antibody-based biologics – protein half-life

IgG and HSA: recycling by neonatal Fc-receptor

PK considerations (mouse)• Antibody domain has completely different PK profile compared to IgG or Fc-fusion

• Data used for simulation: bivalent VHH (Hoefman et al., 2015; Zhang et al., 2016): daily i.v. dosing

• Fc-fusions have similar PK profile to normal IgG, but slightly accelarated clearing• Data used for simulation: human IgG1 based biologics (Unverdorben et al., 2016): 1 i.v. dose/week

0

20

40

60

80

100

120

0 24 48 72 96 120 144 168

µg/m

l

hours

PK based on literature data

AFN IgG Fc-fusion(VHH)2

i.p. injection≈ 8-12h delay

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Antibody-based biologics – protein half-life

• Half-life extension technologies with clinical experience• PEGylation (few days to 1 week in man)

• large hydrodynamic size of 10-40 kD PEG• requires chemical coupling on e.g. N-terminus or (engineered) free cysteine

• (human) serum albumin (1-2 weeks in man)• fusion protein: link with SA or link with SA binding domain antibody

• human SA not recycled in mice, SA binding domain antibody needs to be X-reactive

• Fc (1-3 weeks in man)• comes with bivalency and effector functions (can be engineered out)• recombinant expression typically in mammalian cells

Half-life extension may (will) affect potency and solubility of your drug

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Antibody-based biologics – other POI

• Effector functions or knock-out thereof• add Fc or mutate it

• Immunogenicity• Increase homology with human homologue• Test in silico or in vitro (T-cell epitopes)

• Solubility• very high for s.c.

• …

• BispecificsMultispecifics• 2 or more targets• Introduce cell-specificity: bispecific with cell-type specific targetting antibody linked to target

of interest antibody

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