Site-specific Conjugation for the Advancement of New Linker-Payloads L. Nathan Tumey [email protected] Pfizer Proprietary │ 1
Site-specific Conjugation for the Advancement of New Linker-Payloads
L. Nathan [email protected]
Pfizer Proprietary │ 1
Questions we are asking….
• How do we pick a the best site?• What advantages can site mutants bring?• How can we avoid a “combinatorial nightmare”?
Pfizer Proprietary │ 2
The ideal scenario
Biological Stability
Small set of highly preferred sites
“The Universe of Sites”
Pfizer Proprietary │ 3
Pfizer Proprietary │ 4
Overview of Pfizer Site-specific Technology
Site-specific cysteine Transglutaminase-mediated
Ok, so we’ve found some sites
to use: Now what?
Site has little effect on in vitro potency
Pfizer Proprietary │ 5
Pfizer Proprietary │ 6
2010-2011: Site may have impact on PK
A114CE380C*L398CL443CV422CConventional*Control Ab
Antibody/Time(anti-IgG)
ADC/Time(anti-MMAD)
The 380C-mcMMAD has good tAb PK but poor ADC PK. Why??
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PEGS 2011: Genentech presentation
May 8th, 2011
Rapid ring opening
Slow ring opening
Shen, B. et al., Nature Biotech., 2012, 184.
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Lyon R. P. et al., Nature Biotech., 2014, 32(10), 1059-1062
Tumey L. N. et al., Bioconjugate Chem., 2014, 25(10), 1871-1880
Christie, R. J. et al, J. Controlled Release (2015), Ahead of Print.
Using ring-opening to block deconjugation
Blocking deconjugation: Forced ring opening
Blocking deconjugation: Spontaneous ring opening
But is this all really necessary?
Pfizer Proprietary │ 9
443C ADCs are stable – but do not significantly ring open over time in mouse plasma
0 hr
72 hr
HC-DAR1
+H2O
HC-DAR1
+H2O
In source fragmentation
Hours post injection
Con
cent
ratio
n (u
g/m
L)
ADC/Ab = 100%(mouse)
Pfizer Proprietary │ 10
A variety of sites prevent deconjugation ofvc-Aur0101 in plasma
% D
AR lo
ss%
ring
-ope
ning
Stable site, no ring-opening
Stable site, complete ring-opening
Pfizer Proprietary │ 11
Do these site mutants exhibit similar efficacy?
SiteADC Exposure (hr-
ng/mL)388 3680347 4070443 4500334 4500183 5980392 6010
Tum
or v
olum
e (m
m3 )
Days post-dose
Efficacy of vc0101 conjugate in N87 xenograft following a 1 mpk dose of ADC
Maximizing efficacy requires maximizing ADC exposure
Pfizer Proprietary │ 12
Con
cent
ratio
n (u
g/m
L)
Time (hours)
HIC rrt = 1.35(AUC = 28500 mg*h/mL)
HIC rrt = 1.91(AUC = 11000 mg*hr/mL)
Kappa-K183C vc0101 (tAb)Kappa-K183C vc0101 (ADC)Fc-L443C vc0101 (tAb)Fc-L443C vc0101 (ADC)
Site mutants can impact tAb exposure
Rat PK study
Pfizer Confidential │ 13
Does HIC RRT correlate with tAb PK exposure?
05000
1000015000200002500030000
0.9 1.1 1.3 1.5 1.7 1.9 2.1
Rat tAb Exposure (10 mpk)
0
2000
4000
6000
8000
0.9 1.1 1.3 1.5 1.7 1.9 2.1
Mouse tAb Exposure (3 mpk)C-term LCk183C
C-term HC443C
C-term HC443C
C-term LCk183C
Pfizer Confidential │ 14
Site impacts ADC hydrophobicity
TG-F
C
Possible impact of lower hydrophobicity:1) Improved tAb exposure2) Decreased potential for metabolism3) Improved conjugatability
HIC
RR
T
Pfizer Confidential │ 15
Site impacts payload release kinetics
20 minutes
1.40 1.38 1.15 1.64 1.22 1.55 1.71 1.29 1.60 1.78HIC RRT
Pfizer Confidential │ 16
Site impacts payload release kinetics
HIC RRT
% C
leav
age
@20
min
s
Site impacts ADC metabolism
Mouse Plasma (in vitro)
Hinge-conjugate
392C conjugate
334C conjugateIn vivo mouse PKHinge conjugate: 84% acetate loss @ 72h334C conjugate: 5% acetate loss @ 72h
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Site impacts conjugatability: UncialamycinSEC after attempted conjugation to Tras-A114C:
Pfizer Confidential │ 19
Mutant LCMS DAR %Agg % Right shiftedK246C 0.8 1% 0%K334C 1.5 2.6% 0%Q347C 1.4 21% 63%Y373C 1.2 0% 36%S375C 1.8 0.6% 0%
E380C 1.8 0% 100% (only slight)E388C 1.3 0% 68%K392C 2 2.5% 2.5%N421C 1.4 77% 0%L443C 1.8 87% 0%
kap-A111C 0.7 0% 50%kap-K149C 0.9 3% 50%
kap-K183C 1.9 0% 87% (only slight)kap-K188C 1.4 11% 39%
Screening of site-mutants enabled the conjugation of LPs that were otherwise intractable. All 4 uncialamicin LPs were successfully attached to the 392 and 375 sites.
A114C:
Site impacts conjugatability: Uncialamycin
HIC RRT
1.24
1.28
1.87
1.34
1.59
Pfizer Confidential │ 20
Only particular cys-mutants can be successfully conjugated to calicheamicin
%Aggregation
100%
80%
60%
40%
20%
0% 20% 40% 60% 80% 100%
“Conjugatability” of a calicheamicin LP at various sites
DAR
(%Th
eore
tical
)
Pfizer Confidential │ 21
Site-of-attachment dramatically impacts calicheamicin ADC hydrophobicity
HIC
RR
T
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SummarySite of conjugation can impact:• LP deconjugation• LP metabolism• tAb exposure• Rate of linker cleavage• ADC aggregation• ADC hydrophobicity
Optimization of the site-of-attachment is an essential component of new LP discovery efforts. Careful selection of site can solve a variety of problems that arise during LP optimization efforts.
Pfizer Confidential │ 23
Our early view of site mutants:
Biological StabilitySmall set of highly preferred sites
“The Universe of Sites”
Pfizer Confidential │ 24
Multiple “privileged” sites serving as “tools” to solve specific problems Biological Stability
The emerging scenario…
“The Universe of Sites”
Pfizer Confidential │ 25
AcknowledgementsCarolyn LeverettJesse TeskeS. Chetan SukuruJack BikkerMelissa Wagenaar
Pharmacokinetics, Dynamics, and Metabolism Frank BarlettaJo-Ann WentlandTracey ClarkMauricio LealXiaogang (Sean) HanBrian RagoFengping LiCong WeiSteve Hansel
Worldwide MedchemChris O’DonnellEdmund GrazianiChakrapani SubramanyamRussell DushinFrank KoehnBeth VetelinoSujiet PuthenveetilJeff CasavantAndreas MadernaAnokha RatnayakeZecheng ChenMatt DoroskiHud RisleyAlex PorteGary FilzenLudivine MoineDahui ZhouKen Dirico
Global Biologic TechnologiesWill SomersLioudmila TchistiakovaKim MarquetteSadhana JainMark KrebsMadan KatragaddaRita AgostinelliNicole Piche-NicholasRyan JackobekEric BennettAmy TamLaura LinEric SousaTao He
Oncology ResearchHans Peter GerberPuja SapraPavel StropKathy DelariaFrank LoganzoKiran KhandkeManoj CharatiWilliam HuSylvia MustoJudy LucasNadira PrashadEllie MuszynskaJacob Vineberg