Site-specific Conjugation for the Advancement of New...

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”?


The ideal scenario

Biological Stability

Small set of highly preferred sites

“The Universe of Sites”



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



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??


PEGS 2011: Genentech presentation

May 8th, 2011

Rapid ring opening

Slow ring opening

Shen, B. et al., Nature Biotech., 2012, 184.


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?


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)


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


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


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


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


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


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



Site impacts conjugatability: UncialamycinSEC after attempted conjugation to Tras-A114C:


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


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

)


Site-of-attachment dramatically impacts calicheamicin ADC hydrophobicity

HIC

RR

T


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.


Our early view of site mutants:

Biological StabilitySmall set of highly preferred sites



Multiple “privileged” sites serving as “tools” to solve specific problems Biological Stability

The emerging scenario…



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

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