Third Long-Acting Injectables & ImplantablesConference, La Jolla, CA 6-7 February 2020

Wim Jiskoot

Third Long-Acting Injectables & Implantables Conference, La Jolla, CA6-7 February 2020

Long-acting injectables & implantables: immunogenicity concerns

Acknowledgements

Utrecht UniversityA…Z of the teamBernard MetzCarmen ArigitaDaan CrommelinEnrico MastrobattistaGert StormHerman VromansHuub SchellekensKarin WensinkMarjan FretzMaaike van SlootenMaryam AmidiMelody SauerbornMies van SteenbergenNatasa JovanovicNiels HagenaarsSuzanne HermelingWim Hennink

RIVM/NVI/IntravaccA…Z of the teamCoen BeuveryGideon KerstenHilde VrielingJanny WestdijkPeter SoemaRene RaevenThomas Michiels

Leiden University/LUMCA…Z of the teamAhmad SediqAlexander KrosAna SilvaAndrea HaweBasak KükrerBram SlütterChristophe BarnierEleni VarypatakiElly van RietFerry OssendorpFrank StaalJanine van DuijnJeroen BussmannJeroen HeutsJoke BouwstraJuha MonkareKarin HoogendoornKarin PikeKoen van der MaadenMarc Sutter

Coriolis PharmaA…Z of the teamAdam GrabarekAndrea HaweAriadna MartosDaniel WeinbuchMichael WiggenhornTim Menzen

Rest of the worldAnyone I forgot…Camilla FogedChristian SchöneichDavid VolkinDennis ChristensenGeert-Jan WitkampGerhard WinterHanns-Christian MahlerJanos SzebeniJared BeeJohn CarpenterJohn den EngelsmanLinda NarhiMarco van de WeertMark SchenermannMiao YuRuss MiddaughTed RandolphTudor ArvinteWolfgang Friess

Miranda van BeersMyrra CarstensNaomi BennePauline MeijReza NejadnikRiccardo TorosantucciRobert PooleRomain LebouxStefan RomeijnSuzanne BalVasco Filipe

Immunogenicity of a long-acting injectable: adalimumab (Humira®)

Bartelds et al. Ann Rheum Dis 66: 921-926 (2007)

Immunogenicity: definition

The ability of a substance(e.g., antigen, vaccine, or drug delivery system) to

elicit an immune response

Antibodies

ADCC(antibody-dependent cell-

mediated cytotoxicity )

Cytotoxic (CD8) T cells

CDC(complement dependent

cytotoxicity)

Hypersensitivity

Innate immunity (e.g., clearance by macrophages)

Cytokinestorm

Helper (CD4) T cells

Complement activation

Other

Foreign body reaction

Immunogenicity: why bother?

Immune responses can be

• Good (life-saving, curing)

• Bad (compromising efficacy; side effects)

• Ugly (life-threatening)

Why are you using liposomes for both

drug delivery and vaccine delivery, but

only look for antibodies when the

purpose is vaccine delivery?

Wim Jiskoot, 1985

Particulate drug delivery systems versus vaccinesOur current vaccines are based on particlesVaccine category Vaccine examples Particle size category

Live bacteria Salmonella Micron (~ 1 µm)

Inactivated bacteria Whole cell pertussis Micron (~ 1 µm)

Live viruses Oral polioMeasles-mumps-rubellaNasal influenza

Submicron (~ 30-300 nm)

Inactivated viruses Inactivated polio vaccineInactivated influenza vaccine

< 100 nmSubmicron (~ 200 nm)

Virus like particles Hepatitis B < 100 nm

Split and subunit vaccines Influenza Submicron(aggregates, ~ 300 nm)

Alum-adsorbed antigensMF59 adjuvanted antigens

Diphtheria, tetanusInfluenza

Micron (low µm range)Submicron (~200 nm)

Our current vaccines are based on particles: why?

• Nanoparticles (and small microparticles) loaded with antigens are much more efficiently taken up by antigen-presenting cells (APCs) than antigens free in solution

• Uptake by APCs is the first essential step towards (but not a guarantee for) an adaptive immune response

• Particle uptake by APCs and an immune response thereafter strongly depend on particle properties, e.g.:

• size• composition• shape• surface characteristics (charge, chemistry)• rigidity

PLGA-based adjuvants: size matters!

Joshi et al. AAPS J 15: 85-94 (2013)

Size-dependent uptake of fluorescently labeled protein by APCs (dendritic cells)

Size-dependent antibody response against the protein (ovalbumin)

More than particle size alone…

DSPC:DSPG

DSPC:DSPG:C

HOL

DSPC:DPPG:C

HOL

DPPC:DPPG:C

HOL

DSPC:DOPG:C

HOL

DOPC:DOPG:C

HOL

DOPC:DOPG

0

2000

4000

6000

YM (k

Pa)

DLS: Z-average diameter: 138 - 177 nm; polydispersity index < 0.15

Decreasing rigidity

Benne et al., J Control Release 318: 246-255 (2020)

Liposome rigidity measured by atomic force microscopy

Decreasing rigidity

Antigen-specific regulatory T-cell responses in vitro

Immunogenicity of antigen-loaded anionic liposomes depends on particle rigidity

Kijanka et al. J Pharm Sci 107: 2847-2859 (2018)

Stress protocol: pH 4.6, 65˚C, 30-60 min + stirring (700 rpm, 30 min)

Aggregation FractionationMurine monoclonal IgG1

Stressed monomers

Oligomers

Nano-sized aggregates

Micron-sized aggregates

BALB/c

Anti-drug antibody detection

How about protein aggregates?

Immunogenicity of mAb aggregates: size matters!

Kijanka et al.J Pharm Sci 107: 2847-2859 (2018)

ADA day 65

Unstres

sed

pH Dimer

pH Monomer

Temp Dim

er

Temp M

onomer

UV Dimer

UV Monomer

Supernata

nt 30123

6

8

10

12

S/B

Kijanka et al.J Pharm Sci. 109: 730-738 (2020)

Why bother if your system is out of the critical sizerange?

– Particles may form (e.g., protein aggregates)

– Particles may be shed from larger systems and devices (e.g., fragments of biodegradable delivery systems; protein aggregates leaching from delivery systems, etc.)

– Immune cells, such as macrophages and T-cells, can attack larger systems (e.g., implants)• Cf. rejection of whole organs following transplantation

• Inflammatory response against implantable biomaterials

• Foreign body reaction

Foreign body reaction to biomaterials

Adapted from Taraballi et al. Adv Healthcare Mat 7: 1800490 (2018)

Immunogenicity concerns with liposomes

Potential clinical implications:• Unpredictable pharmacokinetics and

drug release

– Reduced efficacy

– Increased toxicity

• Hypersensitivity reactions

– Potentially life-threatening

Liposomal drug delivery system

Antibody formation

Complement activation

Jiskoot et al. Pharm Res 26: 1303-1314 (2009)

Long-circulating liposomes – rapid clearance upon repeated intravenous administration

Biodistribution study:• 99mTc labeled PEG-liposomes• IV administration to rats• Dosing interval of 1 week

Results:• Long circulation time after

IV injection• Accelerated blood

clearance after repeated IV injection (ABC-effect)

Carstens et al., in: Liposome Technology (G. Gregoriadis, Ed.), p. 79-93 (2006)

Blood clearance study:• Antibody-grafted PEG-liposomes • 67Ga labeled liposomes• Dosing interval of 2 weeks

Results:• Long circulation times after

IV injection• Accelerated blood

clearance after repeated IV injection (ABC-effect)

Immunoliposomes – rapid clearance upon repeated intravenous administration

Harding et al., Biochim Biophys Acta 1327: 181-192 (1997)

Blood clearance study:• First dose: free antibody or

immunoliposomes• Second/third dose: free antibody• Dosing interval of 2 weeks

Results:• Long circulation times of the

antibody• Accelerated blood clearance

after prior IV injection of immunoliposomes

Immunoliposomes – coupling of an antibody to liposomes makes the antibody immunogenic

Harding et al., Biochim Biophys Acta 1327: 181-192 (1997)

Immunogenicity of liposomes: what happens?

Antibody formation– Against the carrier– Against the targeting ligand– Against the drug

Also ‘inert’ PEG can induce antibodies

Anti-PEG IgG and IgM response following a single iv injection of PEGylated liposomes in rats

Ishida et al. J Control Release 122: 349-355 (2007)

Why does this happen?

ANTIGENICCapable of interacting with components of the immune

system

Practically any component- Foreign proteins- Self proteins- Small molecules- Polymers- Liposomes…

IMMUNOGENICCapable of inducing an

immune response

Additional TRIGGER

Immune system responds topathogenic structures

Do liposomes (or other drug delivery systems) contain these triggers?

Triggers of the immune system

1. T-cell epitopes– Present in foreign proteins– Present in self proteins– If presented by antigen-presenting cells, they can activate T-cells,

resulting in B-cell activation and antibody production

2. Pathogen-associated molecular patterns (PAMPs)– E.g., LPS, flagellin, bacterial DNA (CpG)– May be present as impurities or contaminants– Used as adjuvants in vaccines

3. Highly organized antigens– Repetitive array of epitopes– Results in T-cell independent B-cell activation and antibody production

Vos et al. Immunol Rev 176: 154-170 (2000) Bachmann et al. Science 262: 1448-1451 (1993)

Dinitrophenol (DNP)• Small molecule• Effect of coupling to polyacrylamide backbone

No antibodies Anti-DNP antibodies

5-10 nm ~60 nm

No antibodies

Immunogenicity of repetitive epitopes:• Optimum spacing ~ 5-10 nm• Minimum valency ~ 10 epitopes

Antibodies against small molecules

Dintzis et al. PNAS 73: 3671-3675 (1976)

murine TNFalfa• Protein• Effect of coupling to virus-like particles (VLP)

Antibodies against self-proteins

Occupancy (%) 100 50 20 10 No VLPSpacing (nm) 6 8.5 14 19.5

Chackerian et al. J Immunol 169: 6120–6126 (2002)

VLP

Self-protein(TNFalfa)

Foreign protein(streptavidin)

VLP

100% occupancy

50% occupancy

Free TNFalfa(No VLP)

Why does this happen?B-cell stimulation by epitope arrays

Jiskoot et al. Pharm Res 26: 1303-1314 (2009)

Molecules in solution

No B-cell activationNo antibody formation

Repetitive epitopes

Crosslinking of B-cell receptors

B-cell activationAntibody formation

B-cell B-cell B-cell

B-cell stimulation by epitope arrays

= dinitrophenol (linked to polyacrylamide)Dintzis et al., PNAS, 1976Dintzis et al., J Immunol, 1989Sulzer & Perelson, Mol Immunol, 1997

= trinitrophenol (linked to polyacrylamideor dextran)

Mond et al., J Immunol, 1979

= various drug molecules (linked to HPMA)Rihova, Adv Drug Del Rev, 2002

= polymer units, e.g., of bacterialpolysaccharides (used in vaccines)

González-Fernández et al., Vaccine, 2008

MW > 100 kDaAt least 10-20 repeatsInter-epitope distance

5-10 nm

• Examples - polymers

Jiskoot et al. Pharm Res 26: 1303-1314 (2009)

B-cell stimulation by epitope arrays

= phosphatidylcholineSchuster et al., J Immunol, 1979Alving et al., J Lab Clin Med, 1996

= cholesterolSwartz et al., PNAS, 1988Alving & Schwartz, Crit Rev Immunol, 1991Alving et al., Curr Top Microbiol Immunol, 1996

= IgG (self protein)Phillips & Dahman, Immunol Lett, 1995Harding et al., BBA, 1997

= Polyethylene glycol (PEG)Ishida et al., J Control Release, 2006, 2007

Jiskoot et al. Pharm Res 26: 1303-1314 (2009)

• Examples - liposomes:So, the term “immunoliposome” can get a verydifferent meaning than the intended one !

Summary:Relative immunogenicity risk of molecule categories

Molecule category

Molecule presentation form

Soluble, monomeric

High-density multi-epitope array

High-density multi-epitope array + adjuvant

Small molecules - + ++Polymers (non protein) + + ++Self proteins + +++ +++Non-self proteins ++ +++ ++++

adapted from Jiskoot et al. Pharm Res 26: 1303-1314 (2009)

Wrapping up

– The immunogenicity of a molecule not only depends on its nature, but also (or: primarily) on how it is presented to the immune system

– Any molecule can be rendered immunogenic

– You may or may not like immunology; but if you don’t like it, this doesn’t mean it is not there

(adapted from Christian Schöneich)

– The less you study the immunogenicity of your drug delivery system, the less immunogenic you think it is

(adapted from Tudor Arvinte)

– Most drug delivery scientists are working on vaccine delivery; they just don’t realize it

(Tom Anchordoquy)

Thank you!

Mechanism of accelerated blood clearance of PEGylated liposomes (ABC effect)

Mohamed et al. Sci Technol Adv Mat 20: 710-724 (2019)

Mechanism of C activation-related pseudoallergy

Mohamed et al. Sci Technol Adv Mat 20: 710-724 (2019)