1Cleavable linkers inantibody drug conjugates
Literature seminar
2020/01/20
M1 Natsuki Konouecontents
Introduction
What is antibody drug conjugates?
Clinically approved ADC
Mechanism of action

Chemically cleavable linker
Acid cleavable linkers
Reducible disulfide
Cleavage by exogenous stimuli
Enzyme cleavable linkers

Summary2

Introduction

3

Antibody-drug conjugate(ADC)
antibody
the selective delivery system developed against an
antigen associated with a specific cancer cell typechemical linker
The molecular chain throughwhich the payload is attachedto the antibody

Drugpayload
the potent anticancer drug

4Drug〇 high cytotoxicity〇 low production cost× low selectivity to cancer cell× rapid plasma clearance〇 high selectivity to cancer cell〇 long plasma half-lives
× limited cytotoxicity

combination
High cytotoxic and selective therapeutics
with long plasma half-lives

Clinically approved antibody drug conjugates 5Mylotarg (2000, withdrawn in 2010
and reapproved in 2017)
For acute myeloid leukemia(AML)
急性骨髄性白血病Adcetris(2011)
For relapsed or refractory Hodgkin lymphoma andsystemic anaplastic large cell lymphoma (ALCL)
ホジキンリンパ腫、ALK陽性未分化大細胞リンパ腫

Angew. Chem. Int. Ed. 2019, 58, 11206 – 11241

6Kadcyla(2013)
For HER2 positive metastatic
breast cancer
HER2陽性転移性乳がん*HER2: human epidermal
growth factor receptor 2Besponsa(2017)
For relapsed or refractory Bcell precursor acutelymphoblastic leukemia(ALL)
前駆B細胞急性リンパ性白血病●There are almost 100 ADCs in clinical trials
Angew. Chem. Int. Ed. 2019, 58, 11206 – 11241

Mechanism of action

7Chem. Soc. Rev., 2019, 48, 4361-4374

Chemically cleavable linkers

8ADC linkers can be classified as ‘cleavable’ or ‘non-cleavable’
Non-cleavable linkers are mainly effective for the treatment of
haematological cancers or tumors with high antigen expression

Cleavable linkers are generally preferred to non-cleavable linkers
due to their range of applicability

There are 4 types of chemically cleavable linkers••••

Acid cleavable
Reducible disulfides
Cleavable by exogenous stimuli
Enzyme cleavable9

Acid cleavable linkers
Acid cleavable linkers aim to exploit the acidity of theEndosomes(pH 5.5-6.2) and lysosomes(pH 4.5-5.0), whilstmaintaining stability in circulation at pH 7.4
This strategy resulted in clinical success in gemtuzumab
ozogamicin(Mylotarg), inotuzumab ozogamicin(Besponsa)

The linker contains an acidsensitive N-acyl hydrazine linkage
Upon acid catalysis, hydrolyses to aketone and a hydrazide-payload
Besponsa linker showed thathydrazone hydrolysis occurred incirculation at a rate of 1.5-2.0%/day
Bioconjugate Chem.., 2002, 13, 47-58

Other hyrazone-containing linker

10

Phenylketone-derived hydrazine linker

Hydrolysed with t1/2 = 2 days in isolated human and mouseplasma despite much higher stability in pH 7.4 buffer
The specific cause is unclear
The highly variable stability of hydrazones has prevented theirutilityNat. Biotechnol.., 2003, 21, 778-784

Acid cleavable linkers containing otherfunctional groups

11

Carbonate linkers with an alcohol-containing SN-38 payload
Introduction of a p-aminobenzyl(PAB)-spacer boosted theserum stability

Most linker technology has moved away from acid cleavablegroups
The requirement for linkers to distinguish between pH 5.0 and pH
7.4 is difficult and development focuses on other approaches
J. Med. Chem.., 2008, 51, 6916-6926

Reducible disulfides

12Disulfides are the most prominent class of chemicalｌy cleavablemotifs found in ADC linkers
Disulfides are stable at physiological pH but are sensitive tonucleophilic attack from thiols

In contrast to the limited reductive power of blood plasma, thecytosol contains high levels of glutathione(GSH) (1-10 mM)
The oxidative stress associated with tumors leads to elevated GSHlevels

Sterically hindered disulfides
structures of linkers in antibody-maytasinoid conjugates(AMCs)

The rate of reduction of disulfide-linked AMCs by
DTT was measured at 37 C, pH 6.5, using an HPLC
assay to follow the release of maytansinoid
huC242-SMCC-DM1 : 
huC242-SPDB-DM4 : ▲huC242-SPDP-DM4 : ▼huC242-SPDB-DM3 : ■huC242-SPPDM1 : ●Bioconjugate Chem. 2011, 22, 717–727

1314in vivo efficacy of AMCs

A: COLO205 model
B: HT29 model
Using ADC linked with huC242(anti-CanAg) antibody

SPP-DM1 linker was most active
Bioconjugate Chem. 2011, 22, 717–727

a second set of efficacy studies

15

A: COLO205 modelB: HT29 model
Using ADC linked with huC242(anti-CanAg) antibody

In this case, SPDP-DM4 was most active

Bioconjugate Chem. 2011, 22, 717–727

This result arises not from the stability profile but
the increased activity of the DM4 metaboliteDirect disulfide-bonding between engineeredcys residues

16

Without excessive stericprotection around thepayloads, excellent in vivo
mouse plasma stability
The stability was highly
site-dependent
The sterically protected
DM3 payload gave thepredicted rise in stability
over DM1Chem. Sci., 2017, 8, 366–370

Traceless disulfide-carbamate technology

17• Novel self-immolating carbamate linker• This technology allows the benefits of disulfide likers to beapplied to a wider range of potential payloads

Increasing stability to reduction
α-methyl group increased the rateof self-immolation with increasingstability to reduction

Mol. Cancer Ther., 2017; 16, 871-878

Cleavage by exogenous stimuli

18

Advantages of extracellular drug release• Avoidance of any disparity in linker cleavage rates caused byvariable biology across patients• The ADCs can be effective when extracellular concentrations ofendogenous triggers are insufficient for payload release19

Thioether-containing linker

Chem. Sci., 2018, 9, 4185-4189

Under exposure to Pd0, this liker releases an amine-linked payload
In the presence of the [Pd(COD)Cl2] , the ADC was potent

The concentration of Pd required fro efficient decaging was non-toxic
in kidney cells, but it is unlikely to be tolerated in vivo

20Trans-cycloocttene linker
Diels-Alder reaction with an externally administered tetrazine
Releasing an amine-containing payload in vivo

The tetrazines with fast kinetic
profiles were unsuitable for use on
their own, due to their rapid plasma
clearance rate(t1/2≈1 min.)partially proved this problem byemploying a pegylated 3-methyl-6trimethylenetetrazine linked to achelated lutetium(III) species
Instability of the trans-cyclooctenDox conjugate to alkene isomerism
was observed, but this adverse
effect on the therapeutic window
was not severeNat. Commun.., 2018, 9, 1484

Enzyme cleavable linkers• Dipeptide-containing linkers• Glycosidase-cleavable linkers• Phosphatase-cleavable linkers

21

Dipeptide-containing linkers
Dipeptide-containing linkers are present in the majority of ADCs
Previously known lysosome cleavable tetrapeptides
Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu were unsuitable forprodrug or ADC applications due to their slow release kinetics,
hydrophobicity and complexity
The carboxydipeptidase activity of cathepsin B enables tocleave dipeptide linkers

22

23

Citrulline

General trend• A hydrophilic residue at P1 is required and hydrolysis rates increase with basicity• Citrulline(Cit) is isoelectronic with Arg, and was preferred to Arg due to syntheticease• The hydrophobic residues Phe, Val and Ala at P2 enable cleavage by cathepsin B
with plasma stability
Due to the steric bulk of the Dox payload, a spacer unit was required for enzymatic
activity
A p-aminobenzyl carbamate(PABC) linkage was used as a self-immolative spacerDipeptide-containing ADCs
More selective and potent than acid-cleavable hydrazonecontaining ADC
Phe-Lys-PABC and Val-Cit-PABC containing linkers weresuccessfully applied to ADCs
When incubated in isolated human plasma, they exhibited long
half-lives, but in mouse plasma the conjugates were less stable
The enzyme responsible for the instability was not cathepsin but
carboxyl esterase 1C(Ces1C)24

Improving the mouse plasma stability of Val-Cit

25

The addition of hydrophilic P3
residues at the N-terminus
increased the mouse plasma
stability of the ADC, without
negatively affecting cathepsin
B-cleavage

Tripeptidic linkers

26

Tripeptidic linkers with more hydrophilicity at P3 to eliminate
cleavage by Ces1C and provide improved aqueous solubility
P3 = Ser gave only slightly increased stability and P3 = Lys
reduced stability over the native Val-Cit, the use of acidic Gluand Asp residues dramatically increased linker stability, withGlu slightly outperforming Asp

Nat. Commun.., 2018, 9, 2512.

Approach to specifically target cathepsin B
Removal of the P1–P2 amide bond, as well as replacing
the P2 residue, eventually settling with the cyclobutane1,1-dicarboxamide (cBu) moiety
The cBu-Cit moiety is more selective towards cathepsin
B than Val-Cit and does not require other proteases inorder to effectively release the payload

J. Med. Chem.., 2018, 61, 989-1000.

27

The Val-Ala motif

28

the Val-Ala moiety has also emerged as an effective proteasecleavable group
In the case of Val-Ala, 7.4 drugs per mAb were loaded without
significant aggregation(>10%)
Val-Cit-containing linker could not reach a DAR >4 because ofexcessive aggregation and precipitation

Med. Chem. Lett.., 2006, 16, 358-362.

Glycosidase-cleavable linkers

29

β-Glucuronidase-cleavable linkers
β-Glucuronidases are hydrolytic lysosomal enzymes in the glycosidase class thatcatalyse the breakdown of β-glucuronic acid residues in polysaccharides
Attachment to the antibody was achieved by substitution of an amide bondortho- to the enzyme-cleavable groupCrucially, the conjugates were highly stable in isolated rat plasma, with an
extrapolated half-life of 81 days, far superior to the dipeptidic linkers

Bioconjugate Chem. 2009, 20, 1242–1250

Novel linker releasing alkyl alcohol-linked payloads

30

a methylene-alkoxy group was added to the PAB spacer to allow therelease of alkohols

N, N-dimethyl ethyl group was stable at pH 7.4 and efficiently
eliminated the payload upon enzyme hydrolysis

Angew. Chem. Int. Ed., 2016, 55, 7948-7951

31β-Galactosidase-cleavable linkers

The enzyme is analogous to β-glucuronidase in its hydrolytic activity,
but instead hydrolyses β-galactoside.
The authors found that this type of linker, when used withtrastuzumab and MMAE, was more potent than the Val-Cit-PABC
analogueEur. J. Med.. 2017, 142, 376-382

Phosphatase-cleavable linkers

32

Lysosomal acid pyrophosphatase and acid phosphatase
are enzymes that hydrolyse pyrophosphates and terminal
monophosphates respectively to their parent alcohols in
the lysosome
Targeting these enzymes for drug release offers twodistinct opportunities• the substrates are naturally highly hydrophilic• alkyl alcohol payloads can be released

Dipeptide-phosphate-containing ADC
Upon lysosomal proteolysis of the Val-Cit group, the PAB unit
was able to eliminate a terminal monophosphate
The terminal phosphate was hydrolysed by acid phosphataseupon elimination, releasing an alcohol payload
Plasma stability studies were limited to only six hours,
insufficient for the lifetimes associated with ADCs

Bioconjugate Chem. 2016, 27, 2081−2088

33

Pyrophophate-containing ADCs

34

The linkers were hydrolysed in lysosomal extract media, firstreleasing the monophosphateappendaged alcohol payload, then theunfunctionalised glucocorticoid drug

Slow drug release was improved by introducing an acetal spacer

J. Am. Chem. Soc., 2016, 138, 1430-1445

Summary35

 Suitable linker technology at the payload attachment site isrequired for safe and efficacious ADCs
 Cleavable linkers are the preferred choice in the treatment ofcancer due to the importance of bystander killing and payload
cytotoxicity
 Acid cleavable ADC linkers were initially promising but stability
requirements has reduced their value The vast majority of current ADCs are disulfide or dipeptide
technologies due to their ability to distinguish between plasma
and target cell conditions

36

 Non-dipeptide based enzyme-cleavable linkers have not yet
been clinically validated despite their encouraging
preclinical results Glycosidase- and phosphatase-cleavable linkers haveexhibited excellent solubility and stability

Appendix

37

Ozogamicin
N-acetyl calicheamicin γ1Ⅰ derivative
DNA cleavage activity

Doxorubicin
anthracycline antibiotics
DNA intercalational compound

Vedotinmonometyl auristatin E ; MMAE
microtubule inhibitor

Dexamethasone, Fluticasone
Steroidal Anti-Inflammatory Drugs(SAIDs)

Emtansine
maytansinoid derivative
Microtubule inhibitor
SN-38
active metabolite of irinotecan
DNA topoisomerase Ⅰ inhibitor
PBD dimer
DNA binding activity

Intracellular disulfide metabolism

38

In the case of DM4 payload,
rapid S-methylation occurs toform the potent thioether
derivative by an intracellular
methyltransferase

Bioconjugate Chem. 2010, 21, 84–92

DM1 payload is not significantly
S-methylated after 8 hours

39

Ces1 is the enzyme responsible for the instability of Val-Cit linkers

Understanding the role of cathepsin B
Cathepsin S is the mostactive enzyme in the family
to a Val-Cit containing ADC40