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Antibody Drug Conjugates: Structure, Safety & Stability
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Transcript of Antibody Drug Conjugates: Structure, Safety & Stability
Antibody Drug Conjugates:Structure, Safety and Stability
Dr Benjamin Young MRPharmS
and Terry Chapman MPharmacol
2
Questions
1. When was the first ADC licensed?
2. What guidance is specifically for ADC stability studies?
3. What do DAR and DOP mean?
4. What amino acids do current ADCs utilize for attachment?
5. What makes ADCs so hazardous?
3
Introductions
• Bath ASU is collaborating with the University of Bath to enable valid stability assessments of ADCs
• This research is partially funded through a government KTP grant
• Go to mabstalk.com and check out the ADC mini-series for more detail
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Overview
1. Introduction
2. Structure
3. Stability
4. Safety
5. The Future
6. Questions
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1. Introduction• Central Concepts• Timeline• Licensed ADCs• Mode of Action• ADCs VS Gold Standards
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1.1 Central Concepts
• Monoclonal antibodies are comprised of four sub-units
• Two pairs of identical heavy and light tertiary proteins
• The chains are joined by disulfide bonds
Heav
yHe
avy
Hea
vyH
eavy
Ligh
tLi
ght
Disulfide Bond
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VH
VL
CH1
CL
CH
2C
H3
• Conserved subunits create the majority of structure in all mAbs
• The variable sub-units enable specific binding
• Antigen binding occurs at either Fab region
• The Fc region recruits the immune system
Fc
Fab
1.1 Central Concepts
Fab
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• An antibody complexed to a pharmacologically cytotoxic toxic agent is antibody drug conjugate (ADC)
• The Antibody is primarilyresponsible for delivery
• The Linker is primarilyresponsible for release
• The Warhead is primarilyresponsible for apoptosis
Payload
Warhead
Linker
1.1 Central Concepts
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1.2 Timeline
1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010
2013 – Kadcyla approved
1982 – Adriamycin-Antibody conjugate demonstratably superior than co-administration
2000 – Mylotarg approved
2011 – Adcetris approved
1958 – (First ADC) Methotrexate linked to cancer targeting antibody
1906 - Dr Paul Ehrlich proposes conjugating antibodies to toxins to target tumour cells
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1.3 Licensed ADCs
• Gemtuzumab ozogamicin, marketed as Mylotarg™ – An anti-CD33 antibody conjugated to calicheamicin
warheads by hydrazone linkers
• Brentuximab vedotin, marketed as Adcetris™– An anti-CD30 antibody conjugated to mono-methyl
auristatin E (MMAE) warheads by dipeptide linkers
• Trastuzumab emtansine, marketed as Kadcyla™ – An anti-HER2 antibody conjugated to DM-1 warheads by
thioether linkers
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54 3
1.4 Mechanism of Action
Taken from: Senter et al., 2012 Nature Biotechnology, 30, 631–637
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1.5 ADCs VS Gold Standards
• ADC toxicity is more directed and specific
• ADCs are harder to develop resistance to
• ADC therapy has better therapeutic outcomes
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1.5 ADCs VS Gold Standards
Increased specificity means wider a therapeutic index
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1.5 ADCs VS Gold Standards
• ADC toxicity is more directed and specific
• ADCs are harder to develop resistance to
• ADC therapy has better therapeutic outcomes
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1.5 ADCs VS Gold Standards
ADCs exert their potent activity more directly
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1.5 ADCs VS Gold Standards
• ADC toxicity is more directed and specific
• ADCs are harder to develop resistance to
• ADC therapy has better therapeutic outcomes
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1.5 ADCs VS Gold Standards
Adapted from from: Hurvitz et al., 2013, JCO, 44:2967-2977
Increased specificity and reduced resistance creates a more effective and tolerable therapy
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2. Structure
• Antibodies• DAR & DOP • Warheads• Linkers• Challenges
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2.1 Antibodies
• Early ADCs and mAbs were murine which raised immune responses
• Chimeric, humanised or human antibodies limit immune responses
• Complete activity is retained in ADCs
• Antibodies are used to direct the warheads and not for their activity
I
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2.2 DAR & DOP
• Drug antibody ratio (DAR) is the number of payloads per antibody
• High DAR = diminished stability and pharmacokinetic profile
• Low DAR = reduced potency
• Distribution of payloads (DOP) is the location of payload attachment
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2.2 DAR & DOP
• Currently attach via cysteine or lysine residues
• Cysteine linked = more restricted DAR & DOP
• Lysine linked = more stable ADC
Attachment
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2.2 DAR & DOP
Licensed ADC medicines are heterogeneous mixtures
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2.2 DAR & DOP
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Handover
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2.3 Warheads
• Early ADCs targeted conventional chemotherapeutic agents
• Current ADCs use warheads too potent for unconjugated use
• Favoured warhead types:– Anti-mitotic agents– Agents that damage DNA– Agents that prevent DNA synthesis
Warhead
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2.3 Warheads
Maytansinoids • Inhibitors of tubulin reorganisation
• Sourced from Maytenus tree bark
• DM1:– Warhead of Ado-Trastuzumab Emtansine– About 1000x more potent than doxorubicin
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2.3 Warheads
Auristatins
• Inhibitors of tubulin reorganisation
• Derived from Dolastatins produced by Dolabella auricularia
• Monomethyl auristatin E (MMAE)– Warhead of Brentuximab Vedotin– Up to 1000x more potent than doxorubicin
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2.3 Warheads
Calicheamicins
• Bind to the minor groove of DNA and cause DNA strand scission
• Isolated from bacteria in the “Caliche pits” in Texas
• Calicheamicin γ1– Warhead of Mylotarg & CMC-544 – 4000 x more potent than
doxorubicin
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2.4 Linkers
Non-CleavableThioether
Enzymatically-CleavableDipeptide
Chemically-CleavableGlutathioneHydrazone
Typi
cal B
ond
Sta
bilit
y
Linker
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2.4 Linkers
Thioether Bonds
• Used in Kadcyla
• High serum stability
• Warhead remains attached to to linker and a lysine residue
[Lysine - Linker - Warhead]
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2.4 Linkers
Dipeptide constructs
• Two most common:– Valine–Citrulline– Phenylalanine–Lysine
• Brentuximab vedotin uses Val-Cit– Linker contains “self-destructing” moiety to
liberate intact MMAE warhead
• Stable in serum
• Cleaved by cathepsin BVal-Cit Phe-Lys
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Brentuximab Vedotin: Release of MMAE ‘Warhead’
Cathepsin B
Breakdown of carbamate to release Drug + CO2
MMAE
Self-destruction of PABC spacer
2.4 Linkers
Hydrazone bonds
• Gemtuzumab ozogamicin
• Acid-labile (pH < 4.5)
• Relatively poor serum stability– 50% warhead release over 48h
• Linker remains attached to warhead or mAb
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3. Stability
• Parental drug stability• Stability studies• Assessing mAb stability• Unique ADC characteristics• Stability of ADCs
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3.1 Parenteral Drug Stability
The stability of a drug is dependent on:• Formulation conditions
– Concentration– Diluent – pH– Container material
• Environmental conditions– Light– Temperature
• In-process compounding procedures– Shaking / filters / equipment
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3.2 Assessing mAb stability
• Assessing the stability of ADCs is complex– Analytical methods must indicate physical, chemical & functional stability
• Ready to use parenteral drugs require extended shelf lives– Currently 2 NHS guidance documents relating to parenteral drug stability– However, these do not provide adequate scope for ADC stability testing
Processes contributing to degradation of mAbs
Native protein
Chemical Stability Physical stability/Aggregation
Oxidation
Deamidation
Hydrolysis
Proteolysis
Conformational Stability(2ry, 3ry, 4ry structure)
Colloidal Stability(multimers, sub-visible/visible particles)
Unfolded states Aggregates
Free energy change Intermolecular interactions
3.3 Stability Studies (biopharmaceutials)
Physical stability• MicroFlow Imaging• Dynamic Light Scattering (DLS)
Physico-chemical stability• SE-HPLC• pH testing• Circular Dichroism (CD)• LC-MS• Protein Separation Analysis (Electrophoresis)
Functional stability• Biological (cell viability) assays
3.3 Stability Studies (biopharmaceutials)
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3.4 ADC Specific
Characteristic Importance Technique/s
Cytotoxic Selectivity Loss of selectivity indicates increased off target activity
Multicellular Bioassay (in vitro)
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3.5 ADC Specific
Characteristic Importance Technique/s
DAR ProfileAverage DAR
Reduced DAR indicates less potency per ADC
Hydrophobic Interaction Chromatography / LC-MS
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3.5 ADC Specific
Characteristic Importance Technique/s
% Naked Antibody Naked antibody acts as an antagonist of the ADC
Hydrophobic Interaction Chromatography / LC-MS
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3.5 ADC Specific
Characteristic Importance Technique/s
Unconjugated species
Unconjugated species may be strongly cytotoxic
Multi-phase monolith chromatography / LC-MS
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3.6 Stability of ADCs
• ADC stability is subject to guidance relating to both biopharmaceuticals and small molecules
• ADC stability is less straightforward– Heterogeneous nature of ADCs poses characterisation challenges
• Need to demonstrate stability of:– Each individual component (mAb, linker and warhead)– Analytical characterisation must confidently demonstrate that
warhead remains attached to mAb
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4. Safety
• Occupational Exposure• Potential hazards to Pharmacy Operators• Ongoing Investigational Work at Bath ASU
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4.1 Occupational Exposure
• Monoclonal antibodies are highly active biological agents– Capable of powerful pharmacological effects
• Occupational exposure associated with 2 main types of risk:– Immunogenic reactions – Biological effects of the mAb engaging with its target antigen
• Toxicity profile of mAbs are poorly characterised– Occupational hazards from long-term, low-level exposure unknown– Hazards extrapolated from side-effects observed at therapeutic doses
• What about ADCs? ?
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4.1 Occupational Exposure
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…
Source: Hospital Pharmacist, Vol. 15, p138, 2008
4.1 Occupational Exposure
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NIOSH List of Antineoplastic and Other Hazardous Drugs in Healthcare Settings, 2014Taken from Center for Disease Control and Prevention Website: http://www.cdc.gov/niosh/docs/2014-138/
4.1 Occupational Exposure
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4.2 ADCs: potential handling risks
• mAbs are proteins – routes of absorption are limited– This is also the case for ADCs… providing they are intact
• Breakdown of ADC and cytotoxic agent release– Contact with stainless steel (e.g. spillage within isolator)– Following exposure to sterilising gases or cleaning agents– Following contact with skin (dermal esterases) – Following ingestion by other routes (e.g. swallowing, eye contact)
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Trastuzumab Emtansine: “weakest link”
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4.2 ADCs: potential handling risks
• mAbs are proteins – routes of absorption are limited– This is also the case for ADCs… providing they are intact
• Breakdown of ADC and cytotoxic agent release– Contact with stainless steel (e.g. spillage within isolator)– Following exposure to sterilising gases or cleaning agents– Following contact with skin (dermal esterases) – Following ingestion by other routes (e.g. swallowing, eye contact)
• ADC / cytotoxic warhead residue cleaning– mAb is water soluble; warhead is organic soluble – Potential contamination risk of subsequent products
• Permeation of warhead through gloves
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Questions we should be asking with regard to ADC handling…
• Are ADCs susceptible to degradation during handling?
• How hazardous are warheads in trace amounts?
• To what extent are they absorbed?
• Are current arrangements adequate?– Containment– Ventilation– Appropriate PPE– Cleaning protocols
4.2 Potential Handling Hazards
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4.3 Research @ Bath ASU
• in vitro toxicity of ADCs (e.g. hepatocytes / keratinocytes)– Is the toxicity of an ADC increased following:
• Spillage on a variety of contact surfaces• Following contact with cleaning agents / sterilants
• Is the increase in toxicity a result of linker breakdown and warhead release?– HPLC characterisation method currently in development– Heterogeneity of ADCs poses significant characterisation
challenge
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Handover
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5. The Future• ADC pipeline in 2014• Increasing specificity• Beyond antibodies• Overcoming efflux• New linkers
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5.1 ADC Pipeline in 2014
Agent Linker Warhead Target PhaseIMMU-110 Hydrazone Doxorubicin CD74 2Mylotarg® Hydrazone Calicheamicin CD33 WithdrawnCMC-544 Hydrazone Calicheamicin CD22 3SAR3419 Disulfide DM4 CD19 2BT-062 Disulfide DM4 CD138 1
BAY-94-9343 Disulfide DM4 Mesothelin 1SAR-566658 Disulfide DM4 DS6 1
IMGN901 Disulfide DM1 CD56 2Kadcyla® Thioether DM1 HER2 LicensedIMGN529 Thioether DM1 CD37 1SGN-75 MC MMAF CD70 1
Adcetris® Peptide (Val-Cit) MMAE CD30 LicensedRG-7596 Peptide (Val-Cit) MMAE CD79b 2CDX-011 Peptide (Val-Cit) MMAE GPNMB 2
PSMA-ADC Peptide (Val-Cit) MMAE PSMA 2ASG-5ME Peptide (Val-Cit) MMAE AGS-5 1IMUU-130 Peptide (Phe-Lys) SN-38 CEACAM5 2
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• MMAF has a charged residue that reduces its ability to cross cell membranes, thereby increasing specificity
5.2 Increasing specificity
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• To overcome issues of solid tumour penetration, smaller portion of mAbs are being used as the delivery system
5.3 Beyond antibodies
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• DM4 is an example of a drug not affected by multi-drug efflux pump 1
5.4 Overcoming efflux
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5.5 Key points
• ADCs are promising therapeutic agents– Targeted therapy + very potent cytotoxicity
• Linker effectiveness is fundamental– Directed warhead release: cornerstone of ADCs– Beast is subdued only when leashed
• Potential OE of pharmacy staff to warheads– Unique chemical nature of ADCs poses questions
relating to safety during aseptic compounding
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Questions
Who was paying close attention?
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Questions
When was the first ADC licensed?
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Questions
In 2000, Mylotarg, was the first ADC licensed.
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Questions
What guidance applies to ADC stability studies?
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Questions
No guidance specifically applies to ADCs“Yellow cover Part 2” is the best starting point
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Questions
What do DAR and DOP mean?
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Questions
DAR stands for drug antibody ratioDOP stands for distribution of payloads
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Questions
Which amino acids are used for attachment?
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Questions
Cysteine and Lysine
NH
CH
C
H2C
O
O
SH
NH
CH
C
H2C
O
O
H2C
H2C
H2C NH2
Cys
LysO Linker
O
N
O
O
LinkerN
O
O Maleimide
NHS ester
mAb residue attachment chemistry
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Questions
What makes ADCs such a hazardous drug class?
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Questions
A lack of knowledge about the safety and stability of ADCs makes them so hazardous
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Thank you
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Questions?
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