The document discusses antibody-drug conjugates (ADCs), which are monoclonal antibodies conjugated to cytotoxic agents. It provides an overview of ADC structure, stability considerations, safety issues, and the future of ADC development. Regarding structure, it describes key concepts like drug-antibody ratio and distribution of payloads. It also discusses the types of cytotoxic agents and linkers commonly used in ADCs. The document outlines guidance for assessing ADC stability and unique stability characteristics. It notes potential occupational safety hazards from ADC handling and describes ongoing research to evaluate ADC toxicity following degradation. Finally, it reviews the current ADC development pipeline and areas of focus, such as increasing specificity and overcoming drug efflux.

1. 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?
2

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. Overview
1. Introduction
2. Structure
3. Stability
4. Safety
5. The Future
6. Questions
4

5. 1. Introduction
• Central Concepts
• Timeline
• Licensed ADCs
• Mode of Action
• ADCs VS Gold Standards
5

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. 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. • 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. 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. 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. 6
5
4 3
1.4 Mechanism of Action
11Taken from: Senter et al., 2012 Nature Biotechnology, 30, 631–637
2
1

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. 1.5 ADCs VS Gold Standards
13
Increased specificity means wider a therapeutic index

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. 1.5 ADCs VS Gold Standards
15
ADCs exert their potent activity more directly

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. 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. 2. Structure
• Antibodies
• DAR & DOP
• Warheads
• Linkers
• Challenges
18

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. 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. 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. 2.2 DAR & DOP
22
Licensed ADC medicines are heterogeneous mixtures

23. 2.2 DAR & DOP
23

24. Handover
24

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. 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. 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. 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. 2.4 Linkers
Non-Cleavable
Thioether
Enzymatically-Cleavable
Dipeptide
Chemically-Cleavable
Glutathione
Hydrazone
29
TypicalBondStability
Linker

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. 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. Brentuximab Vedotin:
Release of MMAE ‘Warhead’
32
Cathepsin B
Breakdown of carbamate to
release Drug + CO2
MMAE
Self-destruction of PABC spacer

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
3. Stability
• Parental drug stability
• Stability studies
• Assessing mAb stability
• Unique ADC characteristics
• Stability of ADCs

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. 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. 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. 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. 3.4 ADC Specific
39
Characteristic Importance Technique/s
Cytotoxic
Selectivity
Loss of selectivity indicates
increased off target activity
Multicellular Bioassay
(in vitro)

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. 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. 3.5 ADC Specific
42
Characteristic Importance Technique/s
Unconjugated
species
Unconjugated species may
be strongly cytotoxic
Multi-phase monolith
chromatography / LC-MS

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. 4. Safety
• Occupational Exposure
• Potential hazards to Pharmacy Operators
• Ongoing Investigational Work at Bath ASU
44

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
4.1 Occupational Exposure

47. 47
…
Source:
Hospital Pharmacist,
Vol. 15, p138, 2008
4.1 Occupational Exposure

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. 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. Trastuzumab Emtansine: “weakest link”
50

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. 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. 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. Handover
54

55. 5. The Future
• ADC pipeline in 2014
• Increasing specificity
• Beyond antibodies
• Overcoming efflux
• New linkers
55

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
• MMAF has a charged residue that reduces its ability to
cross cell membranes, thereby increasing specificity
5.2 Increasing specificity

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
• DM4 is an example of a drug not affected by multi-drug
efflux pump 1
5.4 Overcoming efflux

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. Questions
Who was paying close attention?
61

62. Questions
When was the first ADC licensed?
62

63. Questions
In 2000, Mylotarg, was the first ADC licensed.
63

64. Questions
What guidance applies to ADC stability studies?
64

65. Questions
No guidance specifically applies to ADCs
“Yellow cover Part 2” is the best starting point
65

66. Questions
What do DAR and DOP mean?
66

67. Questions
DAR stands for drug antibody ratio
DOP stands for distribution of payloads
67

68. Questions
Which amino acids are used for attachment?
68

69. Questions
Cysteine and Lysine
69

70. Questions
What makes ADCs such a hazardous drug class?
70

71. Questions
A lack of knowledge about the safety and
stability of ADCs makes them so hazardous
71

72. Thank you
72

73. Questions?
73

74. 74