cl antibody drug conjugates

The Advent ofAntibody-Drug Conjugates

MacMillan Group Meeting4 June 2015

Tracy Liu

Traditional Cancer TherapyThe Double Edged Sword

Anti-cancer treatments should be as aggressive as possible to fully eradicate the tumor...

...but it is precisely this aggressiveness that often causes severe side effects

N

N N

NN

NH

O

CO2H

CO2H

Me

NH2

H2N

methotrexateanti-folate

HN NH

O

OF

5-fluorouracilthymidylate synthase

inhibitor

PtCl NH3

Cl NH3

cisplatinDNA crosslinking agent

Common Chemotherapeutic Agents

■ lack of tumor selectivity - killing of proliferating normal cells

■ requires administration at near the maximum tolerated dosage

■ 99% of cells in a tumor must be killed to achieve complete remission

Traditional Cancer TherapyMaximizing the Therapeutic Window

Limited clinical efficacy of chemotherapeutics is due to an insufficient therapeutic window -

lack of ability to kill enough cancel cells without causing toxicity to normal cells

Current most critical need: Maximization of the Therapeutic Window

Dosage

Maximum Tolerated Dose (MTD)

Minimum Effective Dose (MED)Therapeutic Window

increase MTD = increase selectivity

decrease MED = increase potency

Angew. Chem. Int. Ed. 2014, 53, 3796.

Targeted Cancer TherapyDirect Approaches

Targeting tumor-associated or specific proteins to directly alter their signaling by:

■ direct binding of monoclonal antibody to ■ binding of small molecule drugs to activeantigen expressed on tumor cell surface site of a protein to disrupt normal function

monoclonal antibody

antigen

tumor cell

HN

NH

O

OF

protein

Nature Rev. 2006, 5, 147.

to induce immune responses

Targeted Cancer TherapyIndirect Approaches

that function as a targeting platform for fusion proteins bearing different effector molecules

■ antibody-directed enzyme prodrug therapy

■ seletive activation of a mildly toxic prodrug to a toxic drug at the tumor site through

Reliance on proteins specifically expressed or overexpressed on tumor cell surfaces

monoclonal antibody (fusion protein)

tumor cell

antigen (overexpressed protein)

prodrug

drug

conjugation of an enzyme to a tumor-specific antibody

= enzyme

Nature Rev. 2006, 5, 147.

Targeted Cancer TherapyIndirect Approaches

that function as a targeting platform for fusion proteins bearing different effector molecules

small molecule drug (antibody-drug conjugate)

Reliance on proteins specifically expressed or overexpressed on tumor cell surfaces

toxin (immunotoxins)radionucleotides (radioimmuno conjugate)

immunoregulatory cytokines (antibody-cytokine fusion protein)

effector molecule

Nature Rev. 2006, 5, 147.

monoclonal antibody (fusion protein)

tumor cell

antigen (overexpressed protein)

effector molecule

500

1000

1500

2000

1900-19

10

1911-19

20

1921-19

30

1931-19

40

1941-19

50

1951-19

60

1961-19

70

1971-19

80

1981-19

90

1991-20

00

2001-20

10

2011-present

19961686

787464

471020000

Antibody-Drug ConjugatesA Brief Introduction and History

Anatomy of an Antibody-Drug Conjugate

A. Antibody

B. Linker

C. Small-molecule drug warhead

Number of Publications in Antibody-Drug Conjugate Research1900 - present day

A

B C


Nature Rev. 2006, 5, 147.; Bioconjugate Chem. 2015, 26, 176.

2000199019801970

1975 - advent ofmurine mAB

1987 - first1997 - first therapeutic mAB(unconjugated) - Rituxan

chimeric mAB Genentech/Biogen

2010

2000 - first FDAapproved ADC - MylotargWyeth (Pfizer)

2010 - Pfizerwithdraws Mylotarg

2011 - FDA approvesAdcetrisSeattle Genetics

2010 - FDAapproval ofKadcylaGenentech/Immunogen

mAB = monoclonal antibodyADC = antibody-drug conjugate

Part I.First Generation

Antibody-Drug Conjugates

Part II.Second Generation


Part III.Current Challenges

and Overview ofClinical Performanceand Their Improvementsand Lessons Learned

Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold

Angew. Chem. Int. Ed. 2014, 53, 3796.

■ represents 75% of serum antibodies in humans

The majority of antibody-drug conjugates are built upon the immunoglobulin G (IgG) scaffold

■ protein complex of 4 peptide chains in a Y shape2 identical heavy chains (light purple)2 identical light chains (dark purple)

hinge

antigen binding complementaritydetermining region (CDR)

FC region

region

Fab region ■ Fab = Fragment antigen-binding domain

■ Fc = Fragment crystallizable/constant domainIdeal location for drug conjugation - far from CDR

Domains of a Typical IgG Antibody

Consists of variable (V) and constant (C) domains

Antigen binding CDR domains found at termini

C

V

CH2 domain

Consists of a CH2 domain and a CH3 domain

CH3 domain

Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold Relevant to Drug Conjugation

Bioconj. Chem. 2015, 26, 176.

■ Linking through native cysteine residues

S SS

SS

S

S S


functional groups present on the surface of the antibodyNature of the chemistry between antibody and linker is primarily determined by the naturally occuring

Deconstruction of Antibody-Drug ConjugatesTraditional Methods of Linker Conjugation to Antibody

Bioconj. Chem. 2015, 26, 176.

■ High nucleophilicity of sulfur - naturally high reactivity for conjugation chemistry■ Low abundance of cysteine in primary sequence - easier control of drug to antibody ratio (DAR)

Linking through native Cysteine residues

Pros

■ No free thiols naturally present - partial reduction required■ Selective reduction of the 4 interchain disulfide bridges is most common, but this partial reduction

Cons

can result in a destabilized antibody

S S SH HS

4 interchain disulfide bridges - easier to reduce12 intrachain disulfide bridges - harder to reduce


Linking through native Cysteine residues

HSSH

OH

OH

dithiothreitol (DTT)

PCO2HHO2C

CO2H

tris(2-carboxyethyl)phosphine (TCEP)

HOSH

2-mercaptoethanol

Common disulfide bridge reducing agents

SS

HO

HO PO

CO2HHO2C

CO2H

HOS

SOH

Corresponding oxidized byproducts


Bioconj. Chem. 2015, 26, 176.



S SS

SS

S

S S


functional groups present on the surface of the antibody

Requires reduction to access free thiol

Nature of the chemistry between antibody and linker is primarily determined by the naturally occuring

NH2

NH2

NH2

NH2


Bioconj. Chem. 2015, 26, 176.



■ Linking through native lysine residues






Garbaccio, R. M. Chemistry of Antibody-Small Molecule Drug Conjugates. From Comprehensive Organic Synthesis II, 2014, 9, 438.

■ Naturally nucleophilic functional handle

Linking through native Lysine residues

Pros

■ Greater natural abundance of lysine - control of drug to antibody ratio significantly more difficult

■ Low levels of competitive cysteine and tyrosine conjugation observed in some cases

Cons

H2N OH

OH2N OH

O

HS

H2N OH

O

HONH2

■ No requirement for pre-functionalization prior to conjugation with linker

Lysine Cysteine Tyrosine

~86 lysine residues total spanning all domains~20 accessible for functionalization

NH2

NH2

NH2

NH2


Bioconj. Chem. 2015, 26, 176.



■ Linking through native lysine residues~86 lysine residues total spanning all domains




~20 accessible for functionalization



Bioconj. Chem. 2015, 26, 176.




■ Linking through conserved glycans in CH2 domain

~86 lysine residues total spanning all domains




~20 accessible for functionalizationAsn

OH

FucoseGlcNAcMannose



Bioconj. Chem., 2015, 26, 176.

■ Post-translational glycosylation of N297 (Asparagine) residue provides facile site-specific conjugation

Linking through native Glycan residues

Pros

■ Glycosylation is a heterogenous post-translational modification - difficult to control drug to antibody ratio■ Requires pre-oxidation of vicinal diol moiety on glycan to access the bioorthogonal aldehyde handle

Cons

■ Glycosylation site is in CH2 domain - well removed from antigen binding domain

O HN

ON297NHAc

O

O

OH

MeOH

OH

OHO

O

NHAc

OHOHO

OOH

OHO

O

O

OHOH

OH

O OHOH

OHO

OO

O

NHAc

OHOHO

OH

NHAcHOO

O

O

OH

OHOH

O

OH

OHOH

OO

HO

AcHN

HO OH

HO

CO2

O

HO

AcHN

HO OH

HO

CO2 fully elaborated glycan

fucose

GlcNAcGlcNAc

mannose

GlcNAcgalactosesialic acid

NH2

NH2

NH2

NH2


Bioconj. Chem. 2015, 26, 176.



S SS

S


■ Linking through conserved glycans in CH2 domainPost translational modification glycosylates N297

SS

S S

~86 lysine residues total spanning all domains




~20 accessible for functionalization

Oxidation of terminal sugar furnishes aldehyde

Asn

OH

FucoseGlcNAcMannose


Deconstruction of Antibody-Drug ConjugatesMore on the Antibody

Angew. Chem. Int. Ed. 2014, 53, 3796.

replacement of protein sequences of a mouse antibody with naturally occuring

Advances in recombinant DNA technology have enabled the generation of engineered antibodies

murine mAB chimeric mAB humanized mAB human mAB

mouse CDR human CDR

sequences in humans significantly reduces undesired immune responses

increasing humanization

Fc region of murine mABreplaced with corresponding

human constant domain

only the essentialmurine CDR regions

are retained

First Generation Antibody-Drug ConjugatesTransition from Chemotherapeutics

Adv. Drug Delivery Rev., 1998, 31, 89.

In an attempt to achieve greater selectivity for chemotherapy drugs, first generation antibody-drug conjugates took clinically established cancer drugs as warheads

N

N N

NN

NH

O CO2H

Me

NH2

H2N

methotrexateanti-folate

N

N

OAc

Me

HO CO2MeR

H

MeO

NH

NOH

Me

CO2Me

R = MeR = CHO

vinca alkaloidsanti-mitotic/microtubule agent

KS1/4 - methotrexateKS1/4 - DAVLB

KS1/4 - DAVLBHYDBR96 - doxorubicin

antibody-drug conjugates

O

OH

Survey of 4 clinically evaluated first generation

O

O

O

OOMe OH

OH

MeOH

NH2

OHO

OH

doxorubicinantibiotic

First Generation Antibody-Drug ConjugatesKS1/4S2 - Methotextrate Conjugate

Am. J. Respir. Crit. Care Med., 1994, 150 , 1114.

N

N N

NN

NH

O CO2H

Me

NH2

H2N

O

OH

Lys

KS1/4S2 murine mAB

H2N

methotrexate

LysHN

ONH

O CO2H

NMe

N

N

N

N

NH2

H2N

KS1/4-methotrexate through non-selective EDC coupling

■ significant localization to tumor

■ Phase I clinical trials revealed

■ murine mAB illicited a humananti-murine antibody (HAMA)response in patients

little therapeutic benefit potentiallydue to non-cleavable linker

■ non-cleavable amide linker formed

First Generation Antibody-Drug ConjugatesKS1/4 – 4-Desacetylvinblastine Conjugates

Cancer Res., 1991, 51, 2286.

N

N

OH

Me

HO CO2MeMe

H

MeO

NH

NOH

Me

CO2Me

4-desacetylvinblastine

OO O

Lys

succinic anhydride KS1/4S2 murine mAB

N

N

OEt

HO CO2MeMe

H

MeOO

HN

O

Lys

H2N

KS1/4-DAVLB ■ highly potent in vivo activity with

■ Phase I clinical trials using radiolabeled

■ no increased therapeutic effect

conjugate indicates localization ofdrug to tumor cells

■ patients developed immune responsesto both the antibody and vinca alkaloid

Clin. Pharmacol. Ther., 1990, 47, 36.

■ esterase-labile hemisuccinate linker

greater efficacy than unconjugated drug

First Generation Antibody-Drug ConjugatesKS1/4 – 4-Desacetylvinblastine Conjugates

Cancer Res., 1991, 51, 2286.

N

N

OH

Me

HOMe

H

MeO

NH

NOH

Me

CO2Me

4-desacetylvinblastine derivative

Asn

KS1/4S2 murine mABO

NHNH2

OH

treated with NaIO4

FucoseGlcNAcMannose

N

N

OHEt

HOMe

H

MeO

O

HN

Asn

NH

KS1/4-DAVLBHYD ■ cleavable acid-labile hydrazone linker

■ Phase I clinical trials indicates localization

■ no increased therapeutic effect - premature

drug to tumor cells

■ patients developed immune responsesto both the antibody and vinca alkaloid

■ highly potent in vivo activity withgreater efficacy than unconjugated drug

cleavage of hydrazone

SN

O

O

O

NHN

OHOH

OMe

O

O

OH

OH O

HS

First Generation Antibody-Drug ConjugatesBR96 - Doxorubicin Conjugate

J. Clin. Oncol., 1999, 17 , 478.

O

O

O

OOMe OH

OH

MeOH

NH2

OHO

OH

doxorubicin BR96 chimeric mAB

HN

ON

O

O

H2N

linker with hydrazide and maleimide

BR96 - doxorubicin■ highly potent in vivo activity with

■ advanced to Phase II clinical trials

■ significant gastrointestinal toxicity -

■ 50% of patients developed immune

■ acid-labile hydrazone linker

greater efficacy than unconjugated drug

responses despite chimeric mAB

target antigen expression on normaltissue cells

First Generation Antibody-Drug ConjugatesUniversal Shortcomings and Lessons Learned

Acc. Chem. Res., 2008, 41, 98.

I. Low in vitro potency - conjugation results in decreased cytotoxicity compared to free drug

All four case studies successfully demonstrated localization of drug payload to tumor sites,

■ Limited expression of antigen - tumor cells typically express 1 x 105 receptors/cell

■ Different mechanisms of cellular uptake

■ Need 106 molecules/cell of a moderately potent cytotoxic drug to effect cell kill

■ Conjugated drugs require efficient internalization after binding to antigen

■ Free drugs can diffuse through cell membrane

but in all cases no significant improvement in therapeutic activity was observed...

II. Stability of the linker was inadequately tuned■ Hydrazone linkers were too labile - prone to cleavage prior to cellular uptake■ Amide linker not labile enough - no cleavage to release drug after internalization

III. Antibodies of murine or chimeric origin illicited undesired immune response■ Generation of human anti-murine antibodies■ Rapid clearance of antibody-drug conjugate upon repeat dosing

Improving Antibody-Drug ConjugatesIdeal Characteristics of an Antibody-Drug Conjugate

Acc. Chem. Res., 2008, 41, 98.

A

B C

A. Antibody

B. Linker

C. Small-molecule drug

■ selective for antigens with highcopy numbers (>105/cell) on

■ induces minimal immunogenicresponse

■ homogeneous expression of

■ Stable to circulation in vivo

■ Selectively cleaved only onceinternalized inside target cell

■ Stable to long-term storagein aqueous environments

■ highly potent in vitro - potencyin picomolar range required

■ amenable to introduction offunctional groups for linking

■ water soluble

target cell

■ selective for antigens uniquelyexpressed on tumor cell

antigen on tumor cell

disulfide linkersprotease labile linkers

■ Designed to release drug inits active form (without linkers)

self immolative linkers

■ sensitive to the ideal mechanismof action for specific tumor types



2000199019801970




2010












Second Generation Antibody-Drug ConjugatesMost Commonly Used Drug Payloads

Angew. Chem. Int. Ed., 2014, 53, 3796.

Me ONH

O

O

Me

OH

ClMeO HN

O

OO

ON

MeR

OMe

Me

MeH

maytansanoidsanti-mitotic agent

MeNMe

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

OR2

R1

auristatinsanti-mitotic agent

MeX

OMeOMe

O

S

O

O

OMe

OH

O

OHMeOHO

Me

NH

OMe

OO

HOO

MeO

HNR

OHO

MeSSScalicheamicins

DNA damaging agent

Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates

Angew. Chem. Int. Ed., 2014, 53, 3796.

Me ONH

O

O

Me

OH

ClMeO HN

O

OO

ON

MeMe

OMe

Me

MeH

maytansineanti-mitotic agent

■ 1000 fold more cytotoxic than first generation payloads

resulting in cell arrest in G2/M phase

■ Good aqueous solubility

■ SAR activity indicates that the ester at C3 can be derivatizedfor linker conjugation without impacting drug activity

3

OOH

3

Me

O

maytansinol

OO

3

Me

O

ON

Me

Me

O

SHHO

ONMe

O

SS

Me

Me

1.

2. DTT reduction

■ Binds tubulin to suppress microtubule dynamics,

H2N Lys


Angew. Chem. Int. Ed., 2014, 53, 3796.

Me ONH

O

O

Me

OH

ClMeO HN

O

OO

ON

Me

OMe

Me

MeH

maytansine

3

SH

O

ONS

SNR4R3

O

O

humanized C242 mAB

R1R2

heterobifunctional linker

OO

3

Me

O

ON

Me

Me

O

SS

R3 R4

R1 R2 O

HN

OO

3

Me

O

ON

Me

Me

O

SS

R3 R4

R1 R2 O

HN Lys


Angew. Chem. Int. Ed., 2014, 53, 3796.

Improved mechanism of selective drug release

NH

OSH

O

HN CO2HHO2C

NH2

glutathione

■ glutathione naturally present in high concentration inside tumorcells (millimolar range), but exceptionally low (micromolar range)

disulfide bond reducing agent

in blood stream - selective cleavage upon internalization

■ disulfide linkage is stable under physiological pH

■ stability of the antibody-drug conjugate can be tuned byvarying the sterics of the R groups flanking the disulfide bond

OO

3

Me

O

ON

Me

Me

O

SS

O

HN Lys


Acc. Chem. Res., 2008, 41, 98.

Study on effect of linker stability to therapeutic efficacy

Me

OO

3

Me

O

ON

Me

Me

OS

SNH

O

Lys

OO

3

Me

O

ON

Me

Me

OS

SMe Me

NH

O

Lys

Meincreasingtherapeutic

efficacy

HuC242-DM1

HuC242-DM3

HuC242-DM4

increasedstability in

blood stream

Extreme case - synthesis of a non-cleavable conjugate?

Lys


Angew. Chem. Int. Ed., 2014, 53, 3796.

Me ONH

O

O

Me

OH

ClMeO HN

O

OO

ON

Me

OMe

Me

MeH

maytansine

3

SH

O

ON

O

O

humanized C242 mABSMCC (non-cleavable) linker

OO

3

Me

O

ON

Me

Me

OO

HN

N

O

O

N

O

O

S

H2N

cyclohexane-1-carboxylatesuccinimidyl 4-(N-maleimidomethyl)

HuC242-MCC-DM1


Acc. Chem. Res., 2008, 41, 98.

time (hrs) post administration

% injected dose

plas

ma

conc

entra

tion

(µg/

mL)

tum

or s

ize

(mm

3 )

days post tumor inoculation

■ HuC242-DM4 shows greatest cytotoxicity■ HuC242-MCC-DM1 shows greatest stability(in vivo half life of 134 hrs)

What is the mechanism of action of these maytansanoid antibody-drug conjugates?


Angew. Chem. Int. Ed., 2014, 53, 3796.

Cellular Processing of Disulfide Linked Maytansanoids

NH

LysO

Me

SS

MayR1 R2

X

lysosomalprocessing

NH

O

Me

SS

MayR1 R2

XH3N

CO2

disulfidereduction

HSMay

R1 R2

X

S-methylation

SMay

R1 R2

X

bystander killing

bystander killingMe

NH

LysO

N

O

O

S May

lysosomalprocessing

NH

O

N

O

O

S MayCO2

H3N

poor bystander killingcharged nature prevents diffusion into

neighboring cells

good

excellent


Angew. Chem. Int. Ed., 2014, 53, 3796.

Enhanced Therapeutic Efficacy of Cleavable Disulfide Linkers is Due to Bystander Cell Killing

Second Generation Antibody-Drug ConjugatesAuristatin Antibody-Drug Conjugates

Angew. Chem. Int. Ed., 2014, 53, 3796.

MeNMe

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

dolastatin 10anti-mitotic agent

■ fully synthetic series of highly potent

derivatized for conjugation AND terminal

■ inhibits tubulin-dependent GTP binding andmicrotubule dynamics

■ SAR indicates terminal 3º amine can be

anti-mitotic agents based on SAR studiesHN N

S

Ph

phenethyl amine can be changed without

on dolastatin 10

■ inhibits tubulin-dependent GTP binding and

loss of efficacy

MeNH

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

HN R1

PhR2

monomethyl auristatin E (MMAE) (R1 = Me, R2 = OH)monomethyl auristatin F (MMAF) (R1 = CO2H, R2 = H)

SH


Angew. Chem. Int. Ed., 2014, 53, 3796.

MeNH

iPrHN

O iPr

O

NMe

monomethyl auristatin

HN

HN

H2NO

ONH

ON

O

ONH

O O LG

O

53

mal-caproyl-val-cit-PAB linkerchimeric cAC10 mAB

HN

HN

H2NO

ONH

ON

O

ONH

O O

O

53

N

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

HN Me

PhHOMeS

Mal-caproyl-val-cit-PAB-MMAE


Angew. Chem. Int. Ed., 2014, 53, 3796.


■ Valine-citrulline dipeptide moiety is known to be selectively cleaved by the protease cathepsin B

■ p-aminobenzyl group is self immolating - fragments to release the MMAE drug without any residual groups

HN

HN

H2NO

ONH

ON

O

ONH

O O

O

53

N

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

HN Me

PhHOMeS

Mal-caproyl-val-cit-PAB-MMAEmal-caproyl

valine-citrulline dipeptide

p-aminobenzyl


Angew. Chem. Int. Ed., 2014, 53, 3796.

HN

HN

H2NO

ONH

ON

O

ONH

O O

O

53

N

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

HN Me

PhHOMeS

Mal-caproyl-val-cit-PAB-MMAEmal-caproyl

valine-citrulline dipeptide

p-aminobenzyl

Mechanism of cellular processing of mal-caproyl-val-cit-MMAE conjugate

cathepsin Bmediated peptide

cleavage

H2N

O

O

N

iPrHN

O iPr

O

NMeMe

NH

iPrHN

O iPr

O

NMe

Meself immolation

-PAB-CO2

free MMAEgood bystander killing

SH


Angew. Chem. Int. Ed., 2014, 53, 3796.

MeNH

iPrHN

O iPr

O

NMe

monomethyl auristatin

N

O

O

mal-caproyl (non-cleavable) linkerchimeric cAC10 mAB

N

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

HN

PhMe

Mal-caproyl-MMAF

O

ON

O

O

ON

O

O

SCys OH

O


Angew. Chem. Int. Ed., 2014, 53, 3796.

N

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

HN

PhMe

ON

O

O

SCys OH

O

Cellular Processing of Non-Cleavable Mal-caproyl-MMAF Conjugates

lysosomalprocessing

N

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

HN

PhMe

ON

O

O

S OH

OH3N

CO2

poor bystander killingcharged nature prevents diffusion into neighboring cells

Second Generation Antibody-Drug ConjugatesCalicheamicin Antibody-Drug Conjugates

Angew. Chem. Int. Ed., 2014, 53, 3796.

MeX

OMeOMe

O

S

O

O

OMe

OH

O

OHMeOHO

Me

NH

OMe

OO

HOO

MeO

HNR

OHO

MeSSS

calicheamicinsDNA damaging agent

■ insanely cytotoxic class of anti-tumor

minor groove of DNA, placing ene-

■ too toxic for use as drug warhead -

■ trisulfide converted to disulfide -provides a handle for conjugation

■ aryl tetrasaccharide moiety binds in

antibiotics (0.15µg/kg dose)

diyne warhead within double helix

20 fold less potent N-acetyl analoguedeveloped for applications to ADCs

calicheamicin β1Br - X = Br, R = iPrcalicheamicin γ1Br - X = Br, R = Etcalicheamicin γ1I - X = I, R = Et

N-acetyl calicheamicin γ1I - X = I

O

MeON

Me

O

Me

NHCO2Me


Angew. Chem. Int. Ed., 2014, 53, 3796.

MeI

OMeOMe

O

S

O

O

OMe

OH

O

OHMeOHO

Me

NH

OMe

OO

HOO

MeON

OHO

MeSSS

Me

O

Me

Mechanism of Action of the Calicheamicins

glutathionemediated

sugar O

OHO

S

Michael addn

sugar O

OHO

S

NHCO2Me

NHCO2Me

thiol reduction

NHCO2Mecycloaromatization

(relief of ring strain)

OHO

S

NHCO2Me

Osugar

OHO

S

NHCO2Me

Osugar

DNA

doublestranded DNA

diradical

O2 doublestranded DNA

cleavagecell death

Lys


Angew. Chem. Int. Ed., 2014, 53, 3796.

sugar O

OHO

NHCO2MeSSN

H

O MeMeH2N

hP67.6 humanized mAB

NH2Me

O

OO

ON

O

O

AcBut linker N-acetyl calicheamicin

LysHN

OO

Me

NHN

O Me Me

SS

sugar O

OHO

NHCO2Me

(4-(4'acetylphenyl)butanoic acid)

hP67.6 N-Ac- γ-calicheamicin DMH AcBut


Bioconj. Chem., 2002, 13 , 47.

LysHN

OO

Me

NHN

O Me Me

SS

sugar O

OHO

NHCO2Me

hP67.6 N-Ac- γ-calicheamicin DMH AcBut


■ Linker specifically designed to provide high stability prior to internalization into tumor cells, but is

cleavage via glutathione reduction upon internalization inside cell

only 6% hydrolysis observed at pH = 7.4

readily cleaved once inside the lysosome - hydrazone formed from ketone rather than aldehyde

97% hydrolysis observed at pH = 4.5 at 37ºC over 24 hrs

■ Inclusion of a hindered disulfide moiety in the linker provides a second handle for selective drug



2000199019801970




2010












Current Trends in Research in Antibody-Drug ConjugatesControlling the Drug to Antibody Ratio (DAR) and Achieving Site-Specific Conjugation

Bioconj. Chem., 2015, 26, 176.

Though the underlying protein scaffold is constant in a heterogeneous population of

antibody-drug conjugates, each conjugate has its own set of pharmacokinetic, toxicity,

aggregation, antigen affinity, and drug release properties

Forefront of research in this field currently lies in achieving:

1. Populations of antibody-drug conjugates with a homogenous DAR

2. Site-specific conjugation of drugs on a given antibody

Current Trends in Research in Antibody-Drug ConjugatesMethods of Homogeneous Conjugation Using Natural Antibodies


■ N-terminal conjugation leveraging differences in pKa between terminal and internal amino acids

HN

O

R

NH2N

HO

HO

Me

OPO32-

37 - 50 ºCphosphate buffer pH 6.5

HN

O

O

R

■ Though conjugation via this method is in the antigen binding domain, drug conjugation here doesnot seem to impact antigen recognition and binding

■ Limitation: Reaction sensitive to nature of terminal amino acid - works best for alanine, glycine, aspartateglutamate, and asparagine

■ Resulting ketone product can be easily further functionalized through reaction with oximes bearinga linker or drug

■ Limitation: Some antibodies may not be able to tolerate elevated temperatures required for transamination

Current Trends in Research in Antibody-Drug ConjugatesMethods of Homogeneous Conjugation Using Natural Antibodies


■ Site-specific functionalization of glutamines through enzymatic conjugation

2. H2N–R, transaminase

■ Selectively functionalizes only Q295 residue (flanked by a consensus recognition sequence for abacterial transaminase)

■ Limitation: Requires deglycosylation in the CH2 domain prior to functionalization, which may impact

■ Q295 residue is distant from antigen binding domain

GlnO

NH2

1. Deglycosylation

GlnO

HN R

function and properties of the antibody-drug conjugate

Current Trends in Research in Antibody-Drug ConjugatesMethods of Homogeneous Conjugation Using Engineered Antibodies


engineered Cys at positions

SH

engineered selenocysteines

SeHHSe

(more nucleophilic)with minimal interferenceto antibody function

CHOOHC

aldehyde taggingengineered recognition

sequence for formylglycineselectively at C-terminus

THIOMABs

generating enzyme (FGE)

C-terminal selenocysteine

OO

MeN3

Current Trends in Research in Antibody-Drug ConjugatesMethods of Homogeneous Conjugation Using Engineered Antibodies


N3

H2NOH

Op-azido Phe

O

H2NOH

Opropynyl Tyr

Me

O

H2NOH

Op-acetyl Phe

Protein Engineering to Incorporate Unnatural Amino Acids

Clinically Successful Antibody-Drug ConjugatesFDA Approved Antibody-Drug Conjugates

Angew. Chem. Int. Ed., 2014, 53, 3796.

HN

HN

H2NO

ONH

ON

O

ONH

O O

O

53

N

iPrHN

O iPr

O

NMe

MeMe

OMe O

N

OMe

Me

O

HN Me

PhHOMeS

brentuximab vedotin (Adcetris)

Roche/Genentech/ImmunoGen

FDA approved in 2011 for Hodgkin lymphoma andanaplastic large cell lymphoma (ALCL)

OO

3

Me

OO

N

Me

Me

O

O

HN

N

O

O

S

Me

NH

O

OOH

OMe

Me

HNCl

MeO

ado-trastuzumab emtansine (Kadycla)

Seattle Genetics

FDA approved in 2013 for metastatic breast cancer

Clinically Successful Antibody-Drug ConjugatesFDA Approved Antibody-Drug Conjugates

Angew. Chem. Int. Ed., 2014, 53, 3796.

Wyeth/PfizerGemtuzumab ozogamicin (Mylotarg)

FDA approved in 2000 for acute lymphoblastic lukemia

MeI

OMeOMe

O

S

O

O

OMe

OH

O

OHMeOHO

Me

NH

OMe

OO

HOO

MeON

OHO

Me

O

Me

NHCO2Me

Lys NH

OO

Me

NNH

O Me Me

SS

Withdrawn in 2010 due to toxicity concernsand lack of improvement in patient survival time

Clinically Successful Antibody-Drug ConjugatesAntibody-Drug Conjugates Currently in Clinical Evaluations

Angew. Chem. Int. Ed., 2014, 53, 3796.

Candidate Drug Antigen Lead Indicator Developer/Partner

cl antibody drug conjugates

Documents

tumorspecific antibody

antibodydrug conjugatea

toxic drug

fusion proteins

tumor site throughreliance

tumor cell surface site

specific proteins

targeting platform