Antibody Drug Conjugates

for CancerPresented By: Anvita Jadhav

M. Pharm (IP)

♠ Antibody-drug conjugates or ADCs are a new class of

drugs designed as a targeted therapy for the treatment

of cancer.

♠ ADCs are complex molecules composed of

an antibody linked, via a stable linker with labile bonds,

to a cytotoxic (anticancer) drug.

What are ADCs ?????????????????

ADC timeline

1958

MTX linked

to an

antibody

directed

toward

leukemia

cells

1972

Noncovalent

linked ADC

tested in

animal

models

1975

Covalent

linked

ADCs

tested in

animal

models

1988

Humanized

mAbs

reported

2000

First FDA

approved

ADC

(Mylotarg®)

Schematic illustration of ADC

Mechanism of Action

Binding

ADC binds to

the target

antigen on the

surface of the

tumor cell to

produce an

ADC-antigen

complex

Internalization

Entire

antigen-ADC

complex is

internalized

through

receptor-

mediated

endocytosis

Degradation

ADC is

degraded

inside the a

lysosome to

release the

cytotoxic

drug.

Release

The cytotoxic

drug enters

the

cytoplasm,

where it binds

to its

molecular

target.

Cell Death

The

interaction of

the cytotoxic

drug with

DNA/

microtubules

leading to

apoptosis.

1 2 3 4 5

Mechanism of Action

Antibody

• Antibodies are

immunoglobulins made up

of:

2 Light Chains (identical)

~25 KDa

2 Heavy Chains (identical)

~50 KDa

Fab

Regio

n

Fc

Regio

n

Antigen Binding Sites

Terminology

MAb: Monoclonal Antibodies

From a biology perspective, the design of an effective ADC relies on

Selection of an

appropriate

target antigen

Tumor

expression

levels

Rates of

antigen

internalization

Antibody Fc

format

Target antigens

A successful ADC should target a well-internalized antigen

with low normal tissue expression and high expression on

tumors.

Antigen expression on normal tissues can be tolerated if

expression on vital organs is minimal or absent.

Target antigens for ADCs in preclinical & clinical development

Cancer Target Antigens

Breast CD174, GPNMB, CRIPTO & nectin-4 (ASG-22ME)

Ovarian MUC16 (CA125), TIM-1 (CDX-014) & mesothelin

Lung CD56, CD326, CRIPTO, FAP, mesothelin & GD2

Pancreatic CD74, CD227 (MUC-1) & nectin-4 (ASG-22ME)

Prostate PSMA, STEAP-1 & TENB2

Antigen expression

In general, optimal ADC targets are homogeneously

and selectively expressed at high density on the surface

of tumor cells.

Homogenous tumor expression (although preferred) is

likely not an absolute requirement.

Antigen internalization

Ideally, once an ADC binds to a tumor-associated target,

the ADC–antigen complex is internalized in a rapid and

efficient manner.

Factors influencing the rate of internalization, such as -

Epitope on the chosen target antigen bound by the ADC

Affinity of the ADC–antigen interaction

Intracellular trafficking pattern of the ADC complex

Impact of format

The biological activity of an antibody can depend on

the interaction of its Fc portion with cells that express Fc

receptors (FcRs).

Therefore, selection of the appropriate antibody

format for an ADC is an important consideration.

Classification of linkers

Linkers

Cleavable

linkers

Lysosomal

protease

sensitive

linkers

Acid sensitive

linkers

Glutathione

sensitive

linkers

Noncleavable

linkers

Cleavable linkers

• This strategy utilizes lysosomal proteases, that recognize and cleave a

dipeptide bond to release the free drug from the conjugate.

• Eg.: Valine - Citrulline linker

• This class of linkers takes advantage of the low pH in the lysosomal

compartment to trigger hydrolysis of an acid labile group within the

linker, & release the drug payload.

• Eg.: Hydrazone linker

Lysosomal protease sensitive linkers

Acid sensitive linkers

• This strategy exploits the higher concentration of thiols, such

as glutathione, to release the free drug.

• Eg. Disulphide linker

Glutathione sensitive linkers

Noncleavable linkers

• This approach depends on complete degradation of the

antibody after internalization of the ADC, resulting in release

of the free drug with the linker attached to an amino acid

residue from the mAb.

• Noncleavable linkers has greater stability in circulation

compared with cleavable linkers.

Cytotoxic drugs

• The drugs being used to construct ADCs generally fall into

two categories:

1) Microtubule inhibitors

2) DNA-damaging agents

• The percent of an injected antibody that localizes to a solid

tumor is very small (0.003–0.08% injected dose per gram of

tumor); therefore, toxic compounds with sub-nanomolar

potency are desirable.

ADC Design

Antibody Drug Conjugates

Linker

Antibody

Drug

• Targets a well-characterizedantigen

• Maintainsbinding, stability,internalization,etc.

• Minimalnonspecificbinding

• Cleavable or noncleavable

• Stable in circulation

• Selective intracellular release of drug

• Highly potent

• Non-immunogenic

• Amenable to modifications for linker attachment

Conjugation

Strategies

Chemical

Conjugation

Site-specific

conjugation

Chemical conjugation

• Traditionally, conjugation of linker-drugs to an antibody

takes place at solvent accessible reactive amino acids such as

lysines or cysteines derived from the reduction of interchain

disulfide bonds in the antibody.

Lysine conjugation

• Results in 0–8 conjugated molecules per antibody

• Conjugation occurs on both the heavy and light chain at ~20

different lysine residues (40 lysines per mAb).

• Greater than one million different ADC species can be

generated.

Cysteine conjugation

• Cysteine conjugation occurs after reduction of four inter-

chain disulfide bonds.

• Linker-drugs per antibody can range from 0–8, generating

more than one hundred different ADC species.

Drawbacks of Chemical conjugation

• ADC species differ in

drug load & conjugation

site.

• Therefore, each species

may have differ in in-

vivo PK properties.

• Batch-to-batch

consistency in ADC

production is difficult to

obtain.

Site-specific conjugation

• It has three strategies

1

• Insertion of cysteine residues in the antibody

sequence by mutation or insertion

2• Insertion of an unnatural amino acid

3•Enzymatic conjugation

ADC advantages over Traditional Chemotherapy

Traditional

Chemotherapy ADC

Merits of ADC

• Selective delivery to tumor cells

• Specific binding to target antigen

• Large therapeutic index

• Reduction of adverse effects

• Extended and prolonged circulation half life

Demerits of ADC

• Molecular targets having similar expression may also get exposed

to the drug

• Requires screening of antigen of interest

• Premature release of cytotoxic drug

• Sufficient concentration may not be achieved at target site

Characterization of ADC

Drug to Antibody Ratio (DAR)

Drug Distribution

Size Variant Analysis

Charge-Based Separations

Analysis of Unconjugated Drug

Peptide Mapping Analysis

Approved ADCs

Agent Status Indication Antigen Cytotoxin Linker

Glembatumumab

vedotin

Ph II Advanced

breast

cancer

GPNMB MMAE Cleavable, Val-Cit

Lorvotuzumab

mertansine

Ph II MM, solid

tumors

CD56 DM1 Cleavable, disulfide

BT-062 Ph I MM CD138 DM4 Cleavable, disulfide

ADCs under Clinical Trials

Agent Indication Antigen Cytotoxin Linker

Adcetris®

(brentuximab

vedotin)

HL, ALCL CD30 MMAE Cleavable

Kadcyla®

(trastuzumab

emtansine)

Her2+ metastatic

breast cancer

HER2 DM1 Non-cleavable

ADCs are a new class of

drugs designed as

a targeted therapy for the

treatment of cancer.

ADCs are complex

molecules composed of

an antibody, linker and

drug.

Site-specific conjugation is

preferred over chemical

conjugation due to

decrease heterogeneity.

There are some unresolved

issues such as antibody

affinity, internalization rate

etc

Conclusion

References

• Heidi L. Perez, Pina M. Cardarelli, Shrikant Deshpande, Sanjeev Gangwar,Gretchen M. Schroeder, Gregory D. Vite And Robert M. Borzilleri, Antibody–DrugConjugates: Current Status And Future Directions, Drug Discovery, Pg. No. 1 -13, December 2013

• Peter D. Senter, Potent Antibody Drug Conjugates For Cancer Therapy, CurrentOpinion In Molecular Biology, Pg. No. 1 – 10, 2009

• Pamela A. Trail, Antibody Drug Conjugates As Cancer Therapeutics, Antibodies,2, Pg. No. 113 - 129, 2013

• Siler Panowski, Sunil Bhakta, Helga Raab, Paul Polakis And Jagath Rjunutula,Site-Specific Antibody Drug Conjugates For Cancer Therapy, Mabs 6:1, Pg. No. 1– 12, January/February 2014

• Beverly A. Teicher And Ravi V.J. Chari, Antibody Conjugate Therapeutics:Challenges And Potential, American Association For Cancer Research, Pg. No.6389 – 6397, 2011

• Singh Harsharan Pal, Gullaiya Sumeet, Kaur Ishpreet, Antibody Drug Conjugates:A Leap Ahead In Cancer Treatment, Journal Of Drug Delivery & Therapeutics,4(3), Pg. No. 52 – 59, 2014

• Blaine Templar Smith, Introduction to Diagnostic and Therapeutic MonoclonalAntibodies, Volume 17, Lesson 1, Pg. No. 1 – 34, 2012

• Michelle Arkin and Mark M. Moasser, HER2 directed small molecule antagonists,Current Opinion Investigational Drugs, 9(12) Pg. No. 1264–1276, December2008.

• Aditya Wakankar, Yan Chen, Yatin Gokarn and Fredric S. Jacobson, AnalyticalMethods For Physicochemical Characterization Of Antibody Drug Conjugates,mAbs 3:2, Pg. No. 161 – 172, March/April 2011

• Jun Zhou and Paraskevi Giannakakou, Targeting Microtubules for CancerChemotherapy, Currrent Medicinal Chemistry – Anti-Cancer Agents, Vol. 5, No. 1,Pg. No. 1 – 7, 2005

• France Carrier, Anne Gatignolo, Mary Christine Hollander, Kuan-Teh Jeang, andAlbert J. Fornace, Induction of RNA-binding proteins in mammalian cells by DNA-damaging agents, Cell Biology, Vol. 91, Pg. No. 1554-1558, February 1994

• Sarah Payne and David Miles, Chapter 4 Mechanisms of Anticancer Drugs, Part 1Cell Biology, Pg. No. 34 – 46, 2007

• S. E. Baldus, S. P. Mönig, T. K. Zirbes, J. Thakran3, D. Kothe, M. Koppel, F. G.Hanisch, J. Thiele, P. M. Schneider, A. H. Hölscher and H. P. Dienes, Lewisy antigen(CD174) and apoptosis in gastric and colorectal carcinomas: Correlations withclinical and prognostic parameters, Histology and Histopathology, Pg. No. 503 –510, 2006

• Patrick J. Burke, Peter D. Senter, David W. Meyer, Jamie B. Miyamoto, MarthaAnderson, Brian E. Toki, Govindarajan Manikumar, Mansukh C. Wani, David J.Kroll, and Scott C. Jeffrey, Design, Synthesis, and Biological Evaluation of Antibody-Drug Conjugates Comprised of Potent Camptothecin Analogues, BioconjugateChem., Vol. 20, No. 6, Pg. No. 1242 – 1250, 2009