Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Antibody Drug Conjugates:
Structure, Safety and Stability
Dr Benjamin Young MRPharmS
and Terry Chapman MPharmacol
Questions
1. When was the first ADC licensed?
2. What guidance is specifically for
ADC stability studies?
3. What do DAR a...
Introductions
3
• Bath ASU is collaborating with the
University of Bath to enable valid
stability assessments of ADCs
• Th...
Overview
1. Introduction
2. Structure
3. Stability
4. Safety
5. The Future
6. Questions
4
1. Introduction
• Central Concepts
• Timeline
• Licensed ADCs
• Mode of Action
• ADCs VS Gold Standards
5
1.1 Central Concepts
• Monoclonal antibodies are
comprised of four sub-units
• Two pairs of identical heavy
and light tert...
CH2CH3
• Conserved subunits
create the majority of
structure in all mAbs
• The variable sub-units
enable specific binding
...
• An antibody complexed to a pharmacologically cytotoxic toxic agent is
antibody drug conjugate (ADC)
• The Antibody is pr...
1.2 Timeline
9
1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010
2013 – Kadcyla
approved
1982 – Adriamycin-Antib...
1.3 Licensed ADCs
• Gemtuzumab ozogamicin, marketed as Mylotarg™
– An anti-CD33 antibody conjugated to calicheamicin
warhe...
6
5
4 3
1.4 Mechanism of Action
11Taken from: Senter et al., 2012 Nature Biotechnology, 30, 631–637
2
1
1.5 ADCs VS Gold Standards
12
• ADC toxicity is more directed and specific
• ADCs are harder to develop resistance to
• AD...
1.5 ADCs VS Gold Standards
13
Increased specificity means wider a therapeutic index
1.5 ADCs VS Gold Standards
• ADC toxicity is more directed and specific
• ADCs are harder to develop resistance to
• ADC t...
1.5 ADCs VS Gold Standards
15
ADCs exert their potent activity more directly
1.5 ADCs VS Gold Standards
• ADC toxicity is more directed and specific
• ADCs are harder to develop resistance to
• ADC t...
1.5 ADCs VS Gold Standards
17
Adapted from from: Hurvitz et al., 2013, JCO, 44:2967-2977
Increased specificity and reduced...
2. Structure
• Antibodies
• DAR & DOP
• Warheads
• Linkers
• Challenges
18
2.1 Antibodies
• Early ADCs and mAbs were murine
which raised immune responses
• Chimeric, humanised or human
antibodies l...
2.2 DAR & DOP
• Drug antibody ratio (DAR) is the
number of payloads per antibody
• High DAR = diminished stability
and pha...
2.2 DAR & DOP
21
• Currently attach via cysteine or lysine residues
• Cysteine linked = more restricted DAR & DOP
• Lysine...
2.2 DAR & DOP
22
Licensed ADC medicines are heterogeneous mixtures
2.2 DAR & DOP
23
Handover
24
2.3 Warheads
• Early ADCs targeted conventional
chemotherapeutic agents
• Current ADCs use warheads too
potent for unconju...
2.3 Warheads
Maytansinoids
• Inhibitors of tubulin reorganisation
• Sourced from Maytenus tree bark
• DM1:
– Warhead of Ad...
2.3 Warheads
Auristatins
• Inhibitors of tubulin reorganisation
• Derived from Dolastatins produced by
Dolabella auricular...
2.3 Warheads
Calicheamicins
• Bind to the minor groove of
DNA and cause DNA strand
scission
• Isolated from bacteria in th...
2.4 Linkers
Non-Cleavable
Thioether
Enzymatically-Cleavable
Dipeptide
Chemically-Cleavable
Glutathione
Hydrazone
29
Typica...
2.4 Linkers
Thioether Bonds
• Used in Kadcyla
• High serum stability
• Warhead remains attached to
to linker and a lysine ...
2.4 Linkers
Dipeptide constructs
31
• Two most common:
– Valine–Citrulline
– Phenylalanine–Lysine
• Brentuximab vedotin us...
Brentuximab Vedotin:
Release of MMAE ‘Warhead’
32
Cathepsin B
Breakdown of carbamate to
release Drug + CO2
MMAE
Self-destr...
2.4 Linkers
Hydrazone bonds
• Gemtuzumab ozogamicin
• Acid-labile (pH < 4.5)
• Relatively poor serum stability
– 50% warhe...
34
3. Stability
• Parental drug stability
• Stability studies
• Assessing mAb stability
• Unique ADC characteristics
• Sta...
3.1 Parenteral Drug Stability
The stability of a drug is dependent on:
• Formulation conditions
– Concentration
– Diluent
...
3.2 Assessing mAb stability
36
• Assessing the stability of ADCs is complex
– Analytical methods must indicate physical, c...
Processes contributing to degradation of mAbs
Native protein
Chemical Stability Physical stability/Aggregation
Oxidation
D...
Physical stability
• MicroFlow Imaging
• Dynamic Light Scattering (DLS)
Physico-chemical stability
• SE-HPLC
• pH testing
...
3.4 ADC Specific
39
Characteristic Importance Technique/s
Cytotoxic
Selectivity
Loss of selectivity indicates
increased of...
3.5 ADC Specific
40
Characteristic Importance Technique/s
DAR Profile
Average DAR
Reduced DAR indicates
less potency per A...
3.5 ADC Specific
41
Characteristic Importance Technique/s
% Naked Antibody
Naked antibody acts as an
antagonist of the ADC...
3.5 ADC Specific
42
Characteristic Importance Technique/s
Unconjugated
species
Unconjugated species may
be strongly cytoto...
3.6 Stability of ADCs
• ADC stability is subject to guidance relating to both
biopharmaceuticals and small molecules
• ADC...
4. Safety
• Occupational Exposure
• Potential hazards to Pharmacy Operators
• Ongoing Investigational Work at Bath ASU
44
4.1 Occupational Exposure
• Monoclonal antibodies are highly active biological agents
– Capable of powerful pharmacologica...
46
4.1 Occupational Exposure
47
…
Source:
Hospital Pharmacist,
Vol. 15, p138, 2008
4.1 Occupational Exposure
48
NIOSH List of Antineoplastic and Other Hazardous Drugs in Healthcare Settings, 2014
Taken from Center for Disease Contr...
4.2 ADCs: potential handling risks
• mAbs are proteins – routes of absorption are limited
– This is also the case for ADCs...
Trastuzumab Emtansine: “weakest link”
50
4.2 ADCs: potential handling risks
• mAbs are proteins – routes of absorption are limited
– This is also the case for ADCs...
Questions we should be asking with regard to ADC handling…
• Are ADCs susceptible to degradation during handling?
• How ha...
4.3 Research @ Bath ASU
• in vitro toxicity of ADCs (e.g. hepatocytes / keratinocytes)
– Is the toxicity of an ADC increas...
Handover
54
5. The Future
• ADC pipeline in 2014
• Increasing specificity
• Beyond antibodies
• Overcoming efflux
• New linkers
55
5.1 ADC Pipeline in 2014
56
Agent Linker Warhead Target Phase
IMMU-110 Hydrazone Doxorubicin CD74 2
Mylotarg® Hydrazone Ca...
57
• MMAF has a charged residue that reduces its ability to
cross cell membranes, thereby increasing specificity
5.2 Incre...
58
• To overcome issues of solid tumour penetration, smaller
portion of mAbs are being used as the delivery system
5.3 Bey...
59
• DM4 is an example of a drug not affected by multi-drug
efflux pump 1
5.4 Overcoming efflux
5.5 Key points
• ADCs are promising therapeutic agents
– Targeted therapy + very potent cytotoxicity
• Linker effectivenes...
Questions
Who was paying close attention?
61
Questions
When was the first ADC licensed?
62
Questions
In 2000, Mylotarg, was the first ADC licensed.
63
Questions
What guidance applies to ADC stability studies?
64
Questions
No guidance specifically applies to ADCs
“Yellow cover Part 2” is the best starting point
65
Questions
What do DAR and DOP mean?
66
Questions
DAR stands for drug antibody ratio
DOP stands for distribution of payloads
67
Questions
Which amino acids are used for attachment?
68
Questions
Cysteine and Lysine
69
Questions
What makes ADCs such a hazardous drug class?
70
Questions
A lack of knowledge about the safety and
stability of ADCs makes them so hazardous
71
Thank you
72
Questions?
73
74
Upcoming SlideShare
Loading in …5
×

Antibody Drug Conjugates: Structure, Safety & Stability

Presented by Dr. Ben Young and Terry Chapman at the annual NHS QA Symposium on 15th September 2015.

Antibody Drug Conjugates: Structure, Safety & Stability

  1. 1. Antibody Drug Conjugates: Structure, Safety and Stability Dr Benjamin Young MRPharmS and Terry Chapman MPharmacol
  2. 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? 2
  3. 3. Introductions 3 • 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
  4. 4. Overview 1. Introduction 2. Structure 3. Stability 4. Safety 5. The Future 6. Questions 4
  5. 5. 1. Introduction • Central Concepts • Timeline • Licensed ADCs • Mode of Action • ADCs VS Gold Standards 5
  6. 6. 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 6 HeavyHeavy Disulfide Bond
  7. 7. CH2CH3 • 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 7 Fc Fab 1.1 Central Concepts Fab
  8. 8. • An antibody complexed to a pharmacologically cytotoxic toxic agent is antibody drug conjugate (ADC) • The Antibody is primarily responsible for delivery • The Linker is primarily responsible for release • The Warhead is primarily responsible for apoptosis 8 Payload Warhead Linker 1.1 Central Concepts
  9. 9. 1.2 Timeline 9 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
  10. 10. 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 10
  11. 11. 6 5 4 3 1.4 Mechanism of Action 11Taken from: Senter et al., 2012 Nature Biotechnology, 30, 631–637 2 1
  12. 12. 1.5 ADCs VS Gold Standards 12 • ADC toxicity is more directed and specific • ADCs are harder to develop resistance to • ADC therapy has better therapeutic outcomes
  13. 13. 1.5 ADCs VS Gold Standards 13 Increased specificity means wider a therapeutic index
  14. 14. 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 14
  15. 15. 1.5 ADCs VS Gold Standards 15 ADCs exert their potent activity more directly
  16. 16. 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 16
  17. 17. 1.5 ADCs VS Gold Standards 17 Adapted from from: Hurvitz et al., 2013, JCO, 44:2967-2977 Increased specificity and reduced resistance creates a more effective and tolerable therapy
  18. 18. 2. Structure • Antibodies • DAR & DOP • Warheads • Linkers • Challenges 18
  19. 19. 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 19 I
  20. 20. 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 20
  21. 21. 2.2 DAR & DOP 21 • Currently attach via cysteine or lysine residues • Cysteine linked = more restricted DAR & DOP • Lysine linked = more stable ADC Attachment
  22. 22. 2.2 DAR & DOP 22 Licensed ADC medicines are heterogeneous mixtures
  23. 23. 2.2 DAR & DOP 23
  24. 24. Handover 24
  25. 25. 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 25 Warhead
  26. 26. 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 26
  27. 27. 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 27
  28. 28. 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 28
  29. 29. 2.4 Linkers Non-Cleavable Thioether Enzymatically-Cleavable Dipeptide Chemically-Cleavable Glutathione Hydrazone 29 TypicalBondStability Linker
  30. 30. 2.4 Linkers Thioether Bonds • Used in Kadcyla • High serum stability • Warhead remains attached to to linker and a lysine residue [Lysine - Linker - Warhead] 30
  31. 31. 2.4 Linkers Dipeptide constructs 31 • 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 B Val-Cit Phe-Lys
  32. 32. Brentuximab Vedotin: Release of MMAE ‘Warhead’ 32 Cathepsin B Breakdown of carbamate to release Drug + CO2 MMAE Self-destruction of PABC spacer
  33. 33. 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
  34. 34. 34 3. Stability • Parental drug stability • Stability studies • Assessing mAb stability • Unique ADC characteristics • Stability of ADCs
  35. 35. 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 35
  36. 36. 3.2 Assessing mAb stability 36 • 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
  37. 37. 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)
  38. 38. 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)
  39. 39. 3.4 ADC Specific 39 Characteristic Importance Technique/s Cytotoxic Selectivity Loss of selectivity indicates increased off target activity Multicellular Bioassay (in vitro)
  40. 40. 3.5 ADC Specific 40 Characteristic Importance Technique/s DAR Profile Average DAR Reduced DAR indicates less potency per ADC Hydrophobic Interaction Chromatography / LC-MS
  41. 41. 3.5 ADC Specific 41 Characteristic Importance Technique/s % Naked Antibody Naked antibody acts as an antagonist of the ADC Hydrophobic Interaction Chromatography / LC-MS
  42. 42. 3.5 ADC Specific 42 Characteristic Importance Technique/s Unconjugated species Unconjugated species may be strongly cytotoxic Multi-phase monolith chromatography / LC-MS
  43. 43. 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 43
  44. 44. 4. Safety • Occupational Exposure • Potential hazards to Pharmacy Operators • Ongoing Investigational Work at Bath ASU 44
  45. 45. 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? 45 ?
  46. 46. 46 4.1 Occupational Exposure
  47. 47. 47 … Source: Hospital Pharmacist, Vol. 15, p138, 2008 4.1 Occupational Exposure
  48. 48. 48 NIOSH List of Antineoplastic and Other Hazardous Drugs in Healthcare Settings, 2014 Taken from Center for Disease Control and Prevention Website: http://www.cdc.gov/niosh/docs/2014-138/ 4.1 Occupational Exposure
  49. 49. 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) 49
  50. 50. Trastuzumab Emtansine: “weakest link” 50
  51. 51. 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 51
  52. 52. 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 52 4.2 Potential Handling Hazards
  53. 53. 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 53
  54. 54. Handover 54
  55. 55. 5. The Future • ADC pipeline in 2014 • Increasing specificity • Beyond antibodies • Overcoming efflux • New linkers 55
  56. 56. 5.1 ADC Pipeline in 2014 56 Agent Linker Warhead Target Phase IMMU-110 Hydrazone Doxorubicin CD74 2 Mylotarg® Hydrazone Calicheamicin CD33 Withdrawn CMC-544 Hydrazone Calicheamicin CD22 3 SAR3419 Disulfide DM4 CD19 2 BT-062 Disulfide DM4 CD138 1 BAY-94-9343 Disulfide DM4 Mesothelin 1 SAR-566658 Disulfide DM4 DS6 1 IMGN901 Disulfide DM1 CD56 2 Kadcyla® Thioether DM1 HER2 Licensed IMGN529 Thioether DM1 CD37 1 SGN-75 MC MMAF CD70 1 Adcetris® Peptide (Val-Cit) MMAE CD30 Licensed RG-7596 Peptide (Val-Cit) MMAE CD79b 2 CDX-011 Peptide (Val-Cit) MMAE GPNMB 2 PSMA-ADC Peptide (Val-Cit) MMAE PSMA 2 ASG-5ME Peptide (Val-Cit) MMAE AGS-5 1 IMUU-130 Peptide (Phe-Lys) SN-38 CEACAM5 2
  57. 57. 57 • MMAF has a charged residue that reduces its ability to cross cell membranes, thereby increasing specificity 5.2 Increasing specificity
  58. 58. 58 • To overcome issues of solid tumour penetration, smaller portion of mAbs are being used as the delivery system 5.3 Beyond antibodies
  59. 59. 59 • DM4 is an example of a drug not affected by multi-drug efflux pump 1 5.4 Overcoming efflux
  60. 60. 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 60
  61. 61. Questions Who was paying close attention? 61
  62. 62. Questions When was the first ADC licensed? 62
  63. 63. Questions In 2000, Mylotarg, was the first ADC licensed. 63
  64. 64. Questions What guidance applies to ADC stability studies? 64
  65. 65. Questions No guidance specifically applies to ADCs “Yellow cover Part 2” is the best starting point 65
  66. 66. Questions What do DAR and DOP mean? 66
  67. 67. Questions DAR stands for drug antibody ratio DOP stands for distribution of payloads 67
  68. 68. Questions Which amino acids are used for attachment? 68
  69. 69. Questions Cysteine and Lysine 69
  70. 70. Questions What makes ADCs such a hazardous drug class? 70
  71. 71. Questions A lack of knowledge about the safety and stability of ADCs makes them so hazardous 71
  72. 72. Thank you 72
  73. 73. Questions? 73
  74. 74. 74

×