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Carbon-14 Labelled ADCs & Peptides

Dr Sean L Kitson Investigator of Radiochemistry

sean.kitson@almacgroup.com

www.almacgroup.com

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OBJECTIVE

This Enabling Technology Session will focus on

carbon-14 labelling of ADCs and peptides in

A(D)ME studies

[14C]-Peptide [14C]-ADC

Radiochemistry

4 S.L. Kitson, D. Speed, ‘Carbon-14 Labelled API Manufacturing’, Drug Discovery World, Winter 2012/13, 14, 72-77.

S.L. Kitson, S.J. Jones, W. Watters, F. Chan, D. Madge, ‘Carbon-14 radiosynthesis of 4-(5-chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl)-6-

(trifluoromethyl)-[4-14C]quinolin-2(1H)-one (XEN-D0401), A novel BK channel activator’, J. Label. Compd. Radiopharm; 2010, 53, 140-146.

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Cysteine (-SH)

residues

Lysine (-NH2)

residues

Image Source: C&EN; 2014, 92 (3), 13-21.

S.L. Kitson et al; ‘Antibody-Drug Conjugates (ADCs) – Biotherapeutic bullets’, Chemistry Today, 2013, 31(4), 30-36.

Peptide linker

binding site

• Targeted Therapy

• Mainly towards oncology

• Growing interest within Pharmaceutical

Industry

Potential Advantages:

• Improved targeting of drug

• Increased tolerability of cytotoxic agent

• Re-investigate failed drugs

ADCs – Biotherapeutic bullets

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Ph. I

70%

Ph. III

3%

Ph. II

21%

Approved

6%

Auristatins

55%

Other

9%

Duocarmycins

3% Calicheamicins

3%

Maytansines

30%

SOURCE: Adapted from Roots Analysis, London 2013.

30 ADCs in various stages of clinical development

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Monoclonal Antibody: Specific

for tumour associated antigen on

the surface of target cells. Antigen

has restricted expression in normal

cells

Linker: Attaches the drug to the

antibody: it must be stable in

circulation to release the drug in

the target cell

Drug: Designed to kill target cells

when internalised and released.

Must be applicable to linker

chemistry technology

ADC - Architecture

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Linking

• A cytotoxic drug is attached to an antibody using a chemical linker.

• The combined ADC molecule eg T-DM1, is then injected into the patient.

Binding

• In some breast cancer patients, the tumour cells are covered with lots of receptors called HER2.

• The antibody locks onto one of these receptors, with drug still attached.

Internalisation

• The cell responds by encapsulating the ADC and receptor.

• Once inside the cell, the antibody and receptor begin to break down.

Release

• The cell breaks the antibody into pieces which releases the drug.

• As more copies of the antibody are absorbed, the drug begins to disrupt the cell, eventually killing it.

Source: Adaptation from The New York Times (June 3, 2012)

Mechanism of Action: ADCs

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KADCYLA FDA APPROVED IN 2013

HER2-positive breast cancer

14C – Labelling on ‘Linker’ Site

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ADCETRIS FDA Approved in 2011

Hodgkin lymphoma (HL)

14C – Labelling ‘Drug’ Site

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Specification:

• [14C]-mAb-Protein Conjugate required carbon-14 label on the linker

• Specific Activity of ≥ 1.1 Ci/mg and 4 g of material

CASE STUDY 1:

[14C]-mAb-Protein Conjugate

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Strategy: [14C]-Linker Chemistry

Drug

mAb

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• [14C]-Linker (1 eq) reacted with Protein Drug (via maleimide linkage)

• IPC analysis by HPLC to determine completion of activation

• Reaction temperature critical to minimise degradation

• Unbound [14C]-Linker removed using DF (10 kDa membrane)

Step 1: Drug - [14C]Linker Activation

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• [14C]-Linker-Drug (4.8 eq) conjugated with mAb (via amide linkage)

• IPC analysis by SEC HPLC to determine completion of conjugation

• Product filtered through 0.22 µm filter to reduce bioburden

Step 2: Antibody Conjugation

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• [14C]-mAb-Protein Conjugate purified using HIC chromatography

• Fractions collected and analysed using SEC HPLC

• Salt exchanged using DF and sample concentrated (30 kDa membrane)

• Product filtered (0.22 µm filter) and formulated in pharmacological buffer

Step 3: Purification / Formulation

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• 4.36 g [14C]- mAb-Protein Conjugate obtained

• 21% Radiochemical yield from [14C]-Linker

• Specific activity 1.20 Ci/mg (Gravimetric)

• All customer target specifications were met

• Bacterial Endotoxin levels <0.3 EU/mL

• BioBurden < 1 CFU/0.5mL

S.L. Kitson et al; ‘Antibody-Drug Conjugates: Carbon-14 Labelling Requirements’, Drug Disc. Devel; February 14 (2012) .

Summary: Case Study 1

14C – Labelled Peptides

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• Position of labelled amino acid in peptide structure

(near N-terminus preferred)

• Specific activity required (single or multiple labelled

AA required)

• Type of amino acid (Glycine, Alanine, Valine,

Leucine, iso-Leucine preferred)

• Cost of Amino acid

• Chirality of amino acid

14C – Peptide Synthesis Strategy

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Specification:

• 2 mg [14C]-biotinylated peptide (84-mer)

• S.A. ≥ 300 mCi/mmol

• Terminal amino acid radiolabelled with [U-14C]-L-isoleucine

• Chemical and radiochemical purity ≥ 95 %area

CASE STUDY 2: [14C]-Biotinylated Peptide

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Solid Phase Peptide Synthesis

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Fmoc Deprotection

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Radiolabelling: [14C]-Peptide

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biotin

Radiolabelling: Boc-[14C]-Peptide-Biotin

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Radiolabelling: [14C]-Peptide-Biotin

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Summary: Case Study 2 • 4 mg of [14C]-biotinylated peptide

• HPLC Purity 98.9 %area (RCP), 99.3 %area (UV)

• SA = 338 mCi/mmol

Stability Study:

• Material stable at –20oC over 4 weeks

• 1% drop in RCP at 2oC over 4 weeks

S.L. Kitson, ‘Keeping tags on biomolecules’, Manufacturing Chemist , April, 36-37 (2012).

Specification:

• 240 mg of [14C]-biomolecule

• Specific Activity > 320 mCi/mmol

CASE STUDY 3: [14C]-Biomolecule

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Stage 1: [14C]-Peptide

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Stage 2: PEGylation

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Stage 3: Bio-conjugation

Summary: Case Study 3 • 250 mg of [14C]-biomolecule prepared

• Total Protein 4.4 mg/ml

• Molecular weight identity (SDS Page): equivalent to cold

standard

• Stability issues with intermediate PEG peptide successfully

resolved

S.L. Kitson, T.S. Moody, D.J. Quinn, A. Hay, ‘Carbon-14 Bioconjugation: Peptides and

Antibody-Drug Conjugates’, Pharmaceutical Sciences, Manufacturing & Marketplace Report, May 8 (2013).

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• Targeted therapies (eg ADCs) is a

growing area of interest within the

biopharmaceutical industry

• Increased need for radiolabelled

biomolecules for A(D)ME evaluation

• Carbon-14 Labelling on Linker and Drug

components of the ADC

Enabling Technology

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Department of Biocatalysis & Isotope Chemistry

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Thank you

The hexagonal shapes denote the famous Giant’s Causeway rock in Northern Ireland – these shapes also

connect to the benzene ring used in science