PermaLink Non Conf March 2014

Enabling technologies for
therapeutic development
Click to edit Master title stylePermaLink™
Non-Confidential
PermaLink™ - an executive summary
Glythera is focused on the enhancement of ADC profiles and therapeutic utility through
the development of PermaLink™ – a novel, cysteine-specific linker conjugation platform
technology
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Suitable for controlled addition via available or engineered cysteine residues
Novel chemistry has enabled a family of linkers with highly specific conjugation to
cysteine
The current platform includes 13 linkers, all with specific profiles
Validation has been achieved in a recognised ADC model
Specifically, the PermaLink™ platform is characterised by
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High stability – demonstrably enhanced stability vs maleimide approaches
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Linker stability
ADC stability
Formulation stability
Potential for reduced aggregation during ADC manufacture, leading to more
homogeneous batch production
Low immunogenicity
Glythera are seeking research collaborations and license discussions with ADC-focused
companies for tailored programmes
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SITE-SPECIFIC CONJUGATION
Cysteine-specific conjugation through engineered cysteine residues
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Linker choice – based on optimal combination
of right linker, target antigen & desired payload
Remaining unmet needs relating to linker
conjugation
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The therapeutic window for many ADCs in pre-clinical or
clinical development remains narrow and further
improvements may be required
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Production of ADCs is an area where improvement is
needed
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The specificity of free drug release in cells is a main goal
of all linkers – important for controlling the toxicity of
highly potent payloads
Cleavable linkers
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Acid labile linkers
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ADCs with cleavable linkers against CD19, CD20, CD21,
CD22, CD79b, CD180 showed in vivo efficacy; whereas
non-cleavable linkers were only efficacious against CD22
and CD79b*
Incorporating the right linker will improve efficacy and
reduce detrimental effects such as formation of
aggregates in systemic circulation
Site-specific conjugation can eliminate heterogeneity,
improve stability and increase the therapeutic window
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Designed to be stable at pH levels of blood, but become
unstable and degrade upon contact with pH environment
within lysosomes
Protease cleavable linkers
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Current methods yield heterogeneous mixtures that may
include 0-8 drug species per antibody molecule
Linkers need to be stable in circulating blood, but allow
rapid release of active free drug inside target cells
Optimal linker conjugation technology depends on the
type of linker (cleavable vs non-cleavable) which, in
turn, depends on the target antigen that is chosen
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Conjugation to cleavable and non-cleavable
systems is required
Also designed to be stable in blood/plasma but rapidly
release free drug inside lysosomes in (cancer) cells on
cleavage by lysosomal enzymes
They take advantage of the high levels of protease
activity inside lysosomes and include a peptide sequence
that is recognised and cleaved by these proteases
e.g. with the dipeptide val-cit linkage in Adcetris, which is
rapidly hydrolysed by cathepsin
Disulfide linkers
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Exploits the extracellular reduced glutathione to release
free drug inside the cell
Non-cleavable linkers
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Provide high stability in the blood but are solely
dependent on internalisation, lysosomal delivery and
degradation of the ADC complex to release active drug
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They may not release drug in extracellular space and are
incapable of killing neighbouring (cancer) cells (e.g.
Kadcyla)
*Polson et al; Cancer Res: (2009): 69: 2358-64
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PermaLink ™ is cysteine-specific & highly
amenable to engineered cysteine residues
Site specific conjugation through engineered
cysteine residues
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ADC production using traditional conjugation through
lysine residues or reduction of disulfide bonds results in
heterogeneity in both DAR and location of conjugation
site
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Site-specific conjugation greatly decreases
heterogeneity
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Lysine conjugation results in a DAR of 0-8 and potential
conjugation at around 40 lysine residues/mAb (A)
Conjugation through reduced inter-chain disulfide residues
results in a DAR of 0-8 and potential conjugation at 8
cysteine residues/mAb (B)
Site-specific conjugation utilising 2 engineered cysteine
residues results in a DAR of 0-2 and conjugation at 2
sites/mAb (C)
DAR can be doubled by engineering 4 sites if desired
Engineered cysteine-residue linkage can result in
enhanced therapeutic windows vs conventional cysteine
conjugation
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Engineered cysteine approaches are favourable
vs other conjugation methods
Engineered
Non Natural Amino
Cysteine Residues
Acids
Enzymatic Conjugation
(Glutamine Tag
Glycoengineering, FGE)
Selenocysteine
Antibody engineering
required
Cysteine substitution
Amber stop codon
substitution
Addition of Sec insertion Addition of glutamine tag or adlehyde tag,
sequence
none for glycoengineering or for pre-existing
glutamine tag (eg Gln-295)
Cell-line engineering
required
None
Cell-line expressing
orthogonol tRNA/aaRS
None
Additional reagents
None
required at time of antibody
expression
Cell-line over-expressing formylglycine generating
enzyme (FGE) for FGE method
None for other methods
Non natural amino acids Sodium Selenite
None
Enzymes required for
conjugation
None
None
None
Glycotransferase
Transglutaminase
Conjugation site location
Any location
Any location
C-terminus (other
locations unknown)
Asn-297 for glycoengineering
Pre-existing glutamine tag (Gln-295) or any location for
other methods
DAR
2 or 4
2 or 4
2
2 for glycoengineering
4 for glutamine tag and FGE
Conjugation chemistry
Maleimide
Bromoacetamide
Oxime
Click chemistry
Maleimide
Click chemistry
transamidation
hydrozino-Pictet-Spengler chemistry
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PERMALINK™ PLATFORM
An overview of the platform and results to date
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PermaLink ™ – a novel, cysteine specific linker
technology
Proprietary technology platform
Controlled, stable conjugation platform
PermaLink™ - Linker, conjugation chemistry for;
Highly specific for cysteine amino acids
Controlled conjugation allowing optimal DAR
1.Primary
2. focus
3.
Secondary
focus
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1. Antibody Drug Conjugates
2. Bi-specifics
3. Conjugate vaccines
4. PEGylation
Enhanced stability vs current linkers
Enhanced formulation stability and low
immunogenicity platform
Functional in a range of key conjugated product
classes
Applicability in cleavable & non-cleavable systems
Significantly less cross-linking vs bifunctional
linkers (e.g. Kadcyla mimics)
Glythera’s technology can be used for the targeted addition of cytotoxic payload with increased
stability compared with current technologies
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PermaLink™ meets the profile of an ideal
linker conjugation technology
Target attribute
Key results
Benefits / inference
High cysteine
specificity
In in vitro binding assays with N-acetyl cysteine;
- PermaLink™ binds specifically to cysteine
- (maleimide linkers conjugate non-specifically to non-cysteine containing
proteins)
- PermaLink™ conjugation is not affected by alternative nucleophilic amino
acids
Improved product homogeneity/
improved analytical characterisation
Enhanced linker
stability vs maleimide
Under sustained exposure of 1000-fold free thiol;
- PermaLink™ remains fully bound to cysteine
- PermaLink™ is highly stable and not subject to ring-opening, unlike
maleimide™ linkers
No risk of retro-Michael reaction
resulting in steady state conjugates
Controlled,
predictable
conjugation
- PermaLink™ conjugation steps can easily be manipulated to deliver required DAR
- High monomer purity (>93%) after one purification step
Optimisation of ADC potency as
required
High ADC stability
Under sustained exposure of free thiols in vitro;
- PermaLink™ ADCs are significantly more stable vs maleimide-conjugated
ADCs
- PermaLink ADCs are associated with stable DARs & no de-drugging
Potential for increased delivery of
intact ADC to tumour site
Cell kill activity
When compared with maleimide-ADC control
- PermaLink™-ADC has a comparative cell kill profile
- Proven internalisation and cleavage of PL linkers
Similar in-vitro kinetics and
mechanism of action to current
approved ADCs
Enhanced
formulation stability
When compared with maleimide ADC control;
- PermaLink™ ADCs may be associated with reduced aggregation potential
Improved manufacturing processes –
improved cost of goods through
reduced need for aggregate removal
Low immunogenicity
In T-cell models challenged with free linkers;
- PermaLink™ linkers are associated with minimal risk of immunogenicity
Low risk/ low impact on ADC product
immunogenicity profile
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PermaLink™ linkers have been validated in
a trastuzumab ADC model
Comparison with maleimide linker technology
Glythera has generated a hybrid model alongside in vitro and in vivo assays to assess the effectiveness of the
PermaLink™ technology
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Trastuzumab plus a vit-cit cleavable component + spacer + drug
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Drug activity is conferred only after cleavage of the linker and removal of the self-immolative PABC spacer
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The model has been used to determine in vitro cysteine specificity, stability & internalisation / linker cleavage and cell-kill
capabilities of the PermaLink™ /ADC format
Valine-citrulline cleavable linker
Drug
Maleimido
caproyl spacer
vs PermaLink™
PABC spacer
NB: all appropriate test and reference materials, plus all necessary intermediates
required for production of this PermaLink™ ADC model are scalable to clinical
manufacture
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PermaLink™ conjugation is highly specific
to cysteine
Glythera has demonstrated that PermaLink™ does not conjugate to non-cysteine containing proteins, whilst
maleimide technologies conjugate in a non-specific manner
PermaLink™ has been shown to bind with N-acetyl cysteine (NAC) through the Michael reaction with a free thiol
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RP-HPLC & LC-MS confirm PL13-NAC adduct;
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PermaLink™ conjugation is highly stable
PermaLink™ stability was challenged both whilst conjugating to NAC and up to 72hrs after
conjugation
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Targeted addition of a linker/payload combination is critical in ADC characterisation and in delivering an optimal drug loading
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ADCs will be challenged by free thiols in circulation; albumin competition with conjugation chemistries has been
demonstrated as a key issue in instability and off-target effects in the clinic
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PermaLink™ conjugation was subject to competition from amino acids with nucleophilic side chains (tyr, his and lys)
Amino acid competitive challenge – tyr/his/lys/NAC
Site specific conjugation
Conversion to PermaLink™ (PL-13)-NAC adduct only
PL13-NAC adduct confirmed by LC-MS
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PermaLink™ is not influenced by alternative nucleophilic side chains
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PermaLink™ allows controlled conjugation
for optimal DAR
A range of DAR may be required depending on Mab, cytotoxin and tissue target
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PermaLink™ can be controlled to produce an ADC of characterizable DAR
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Currently developing ADCs with DAR range of 4, 6 & 8
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Comparative cleavable/non-cleavable ADC models with supporting in-vitro/in-vivo assays
Characterisation assays are based on typical CMC analytical methods
L0 H0/L1
H1 H2 H3
We are currently optimising conjugation step to achieve optimal DAR
SEC 93.5 % monomer after single purification step
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PermaLink™ offers enhanced linker stability
No risk of retro-Michael reaction with PermaLink™ associated with maleimide conjugation
technologies
PermaLink™ is resistant to ring opening
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Ring instability can be an issue, resulting in dissociation of mAb from linker/cytotoxic combination
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Dissociation results in reduced tumour site delivery of intact ADC & subsequent off-target effects through (i) albumin
trafficking or (ii) active uptake of free toxin/linker
Linkers can undergo hydrolysis before entering the tumour cell, especially at high pH and temperature. The subsequent ringopened species reduce ADC stability and drug potency
Companies such as GenenTech are now producing analytical methods to quantify ring-opening reactions & characterise the
impact of local environments on specific ring-opening sites
In order to enhance the stability of on-market linkers, some groups (e.g. Seattle Genetics) have encouraged ring-opening
reactions to occur in order to prevent the retro conjugate addition from occurring
PermaLink™
PermaLink™ and Maleimide conjugation
were challenged in vivo in various pH
conditions and for up to 2 days
PermaLink™ was not susceptible to ring
opening at any time throughout the
challenge, whilst maleimide showed clear
evidence of instability
PermaLink™
structure
available
under
confidentiality
pH 9.0
2 days
Maleimide
pH 9.0
2 days
The PermaLink™ stable conjugation platform eliminates the risk of ring-opening events and any
subsequent structural or clinical implications associated with other technologies
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PermaLink™ ADCs are more stable than
maleimide conjugated ADCs
Free amino acid (NAC) challenge to ADC
Linker/toxin instability & generation of free toxin can have significant implications on (i) dosing regimens, (ii)
delivery of intact ADC to tumour site, (iii) associated product efficacy and (iv) off-target effects
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PermaLink™ ADC was challenged in vivo under challenge from 1000-fold concentrations of free thiols for
up to 2 days
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DAR remained stable throughout the period; no evidence of de-drugging at any time
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In contrast, maleimide ADC showed increasing evidence of degradation over time
2
2
0
3
1
4
01
6
8
4
3
6
8
Maleimide ADC
PermaLink™ ADC – no detectable de-drugging, DAR remains stable
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Comparable in vitro activity
PermaLink™ ADC comparable in SKBR3 cell line
 PermaLink™ ADC was tested against the hybrid reference for equivalence in cell kill profile using an accepted release assay
format
 Internalisation and cleavage of the PermaLink™ conjugated linker are confirmed through its comparative cell-kill profile vs
a maleimide control, suggesting similar mechanisms of action for both ADCs
T = 24
T = 72
T = 48
= 78 % response in cell number
= 74 % response in cell number
PermaLink™ ADC
Maleimido caproyl ADC
Trastuzumab
Media
= 30 % response in cell number
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PermaLink™ may be associated with
enhanced formulation stability
Incorporation of PermaLink™ results in a decrease in aggregation potential vs maleimide
technology*
“naked” mAb aggregation is a major concern in the industry and one which impacts on regulatory acceptance
 Incorporation of linker/toxin payloads into ADC formats – i.e. inclusion of hydrophobic technologies – results in an inherent
decrease in product thermal stability when compared to “naked” antibody
 It can also be associated with increased aggregation potential of ADCs
 PermaLink™ was therefore tested against the trastuzumab/maleimide/drug reference for thermal stability and aggregation
potential
Equivalent thermal stability
 No significant change in thermal stability is observed between the maleimide reference and
the PermaLink™ ADC
0.8mg/mL
 Using the PermaLink™ technology is therefore not considered a significant risk - a similar
difference (and therefore risk profile) would be anticipated for the approved products Kadcyla
and Adcetris
Reduced aggregation potential
 The reduced risk of aggregation through the substitution of the PermaLink™ technology in the
trastuzumab model suggests that, even through the inclusion of this hydrophobic chemistry,
the risk of aggregation is returned to a similar level associated with the “naked” mAb
0.8mg/mL
 PermaLink™ should not be associated with any cold-chain supply or fill-finish issues and may
confer a benefit vs other linker technologies in this regard
*according to preliminary data – further validation is ongoing
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PermaLink™ is associated with low
immunogenicity
PermaLink™ does not show independent immunogenic potential
 Inclusion of linker systems into ADC formats have resulted in increased immunogenicity vs naked mAbs presenting a risk in product
approach strategies
“Naked” PermaLink™ was challenged in a proprietary T-cell based assay
 The ‘worst case scenario’ dose was tested – simulating a completely de-conjugated linker (10 mg/Kg dose - ADC with a DAR of 4)
 Samples from 6 patients were collected and subjected to exposure
 DNCB was used as a positive control for immune system stimulation
 TX was used as a negative control (i.e. protection from immunogenicity)
PermaLink™ was not associated with an immunogenicity risk at any dose, including the
maximum (unrealistic challenge) dose of 10 mg/Kg
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PermaLink™ IP overview
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WO 2010/070300 A2 – Functionalising reagents and their uses - filed in China,
Europe, Hong Kong, India, Japan and USA
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First grants expected Q2 of 2014
Further IP in the pipeline
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PermaLink™ development strategy
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Specificity
Stability
Activity
Cleavable linker formats
in vitro immunogenicity
Ongoing
Non-cleavable linker formats
Ongoing
Xenograft model
Ongoing
Additional mAb models
Ongoing
Broader IP
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Glythera are seeking research collaborations to
demonstrate the value of PermaLink™
Glythera are open to various research collaboration models
1.
3rd Party evaluation of PermaLink™ platform
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2.
Technology evaluation – in-house at Glythera or through shared programmes
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3.
Demonstration of PermaLink™ with 3rd Party assets (antibody, including engineered cysteine forms; payload)
to agreed project plans
Assessment of PermaLink™ vs 3rd party ADC assets which utilise ‘established’ linkers
Demonstration of ‘ADC ability’ for 3rd Party antibody / payload assets, utilising PermaLink™
Increased bandwidth through Glythera’s capabilities & expertise
Rapid demonstration of technology strengths through comparative models
Technology development
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4.
Open access project - flexible, low risk approach
Appropriate for “toolbox” approach – deployment into multiple product development streams
Partnered development of the PermaLink™ platform
Co-development of ADC targets
Technology enabled clinical development programmes
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Assuming successful research programmes 1, 2 and/or 3 (above)
Contact information for further discussion;
[email protected]
CEO, Glythera
DD: +44 (0) 191 222 3839
Mob: +44 (0)7910 166 760
[email protected]
Commercial Director
DD: +44 (0)1477 537111
Mob: +44 (0)7802 470108
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