carbon-14 labelled adcs and peptides by sean l kitson
DESCRIPTION
Abstract Guided by a specific monoclonal antibody (mAb), antibody drug conjugates or ADC are a new, emerging, class of drugs able to deliver a drug payload directly to an intended target. This approach has recently been boosted by the U.S. Food and Drug Administration approval of brentuximab vedotin (Adcetris®; Seattle Genetics) to treat Hodgkin’s lymphoma and ado-trastuzumab emtansine (Kadcyla®; Genentech) for metastatic breast cancer. These new biotherapeutic drugs will bring many regulatory issues to the forefront regarding the ADME (Absorption, Distribution, Metabolism and Excretion) profile of each ADC. In this article, the authors discuss this and other important aspects of antibody-drug conjugates.TRANSCRIPT
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Carbon-14 Labelled ADCs & Peptides
Dr Sean L Kitson Investigator of Radiochemistry
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