Targeted quantification of therapeutic monoclonal ...

Targeted quantification of therapeutic monoclonal antibodies in plasma using mass spectrometry 20th June 2018 Dr. Margaux Fresnais

Heidelberg University Hospital Clinical Pharmacology and Pharmacoepidemiology Department (Head: Prof. Dr. Haefeli) Analytical Chemistry Laboratory (Head: Dr. Burhenne) Im Neuenheimer Feld 410, 69120 Heidelberg

Clinical Pharmacology and Pharmacoepidemiology Department

Heidelberg University Hospital – Clin. Pharmacol. Pharmacolepidemiol. - ACL | 20th June 2018 | Dr. Margaux Fresnais

Head

of Department

Prof. Dr. W. E. Haefeli

Analytical Chemistry Laboratory

Dr. J. Burhenne

Molecular Biology Laboratory Prof. J. Weiss

Clinical Research Unit (KliPS)

Prof. G. Mikus

Pharmacoepidemiology Dr. A. Meid

Bioinformatics M. Metzner

Drug Utilization and Drug Safety

Prof. D. Czock

Administration & Organisation

Quality Assurance

Cooperation Unit Clinical Pharmacy

PD Dr. H. Seidling

FIM-Heidelberg First-In-Man

Cooperation with Hospital Pharmacy and Institute for Pharmacy and Molecular Biotechnology

Cooperation partners

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http://www.klinikum.uni-heidelberg.de/Nationales-Centrum-fuer-Tumorerkrankungen.106907.0.html

Analytical Chemistry Laboratory Our missions


Development and application of ultrasensitive analytical assays for the quantification of endogenous compounds, drugs, and their metabolites in human and animal samples from

any biological matrix to address pharmacokinetic, pathophysiological, and toxicological questions.

• Support for clinical drug trials

• Assay validation according to FDA and EMA standards

(e.g. FDA Guidance for Industry: Bioanalytical Methods Validation)

• Quantification of drugs at the action site (drug efficacy monitoring)

• Ultrasensitive drug assays (cancer therapy, pulmonary hypertension, infection, anticoagulation …)

• Therapeutic drug monitoring (routine).

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Analytical Chemistry Laboratory Matrices and targets


Drug quantification

PK/PD studies

Therapeutic Drug Monitoring

Drug efficacy monitoring

Drug distribution

Body fluids

Functionnal sites

Tissues

Biological matrices

•Whole blood

•Plasma

•Urine

• Saliva

•…

•Cells (blood stream)

• Liquor

•Aqueous humor

• Lymphocytes

•Erythrocytes

•…

•Human material from surgery

•Animal material (brain, organ slices,…)

Therapeutic targets Small molecules Peptides Proteins Antibodies

Endogeneous targets Peptides Proteins

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Analytical Chemistry Laboratory Matrices and targets


Drug quantification

PK/PD STUDIES

Therapeutic Drug Monitoring

DRUG EFFICACY MONITORING

Drug distribution

BODY FLUIDS

Functionnal sites

Tissues

Biological matrices

•Whole blood

•PLASMA

•Urine

• Saliva

•…

•Cells (blood stream)

• Liquor

•Aqueous humor

• Lymphocytes

•Erythrocytes

•…

•Human material from surgery

•Animal material (brain, organ slices,…)

Therapeutic targets Small molecules Peptides Proteins ANTIBODIES

Endogeneous targets Peptides Proteins

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Antibody quantification in plasma using MS For what purpose ?


How can we monitor the efficacy of an antibody-based treatment?

Isolation and analysis of the bound fraction to quantify the amount of mAB that reached the target (T-cells): pharmacodynamic study.

Quantification of free mAB in plasma for pharmacokinetic study.

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Antibody quantification in plasma using MS Surrogate peptide approach for mABs


Determination of the signature peptides of an antibody

In silico digestion

Heavy Chain Sequence QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTA

VYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE

VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

ASGITFSNSGMHWVR

AEDTAVYYCATNDDYWGQGTLVTVSSASTK

BLAST* search in databases against every human

protein sequences

* BLAST = Basic Local Alignment Search Tool

Peptides that are unique to the protein of interest

= Candidates for signature peptide

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Bottom-up strategy to quantify signature peptides

Purification of mAB fraction

Biological sample

Immunopurification on Protein A agarose resin

(96-well plate kit)

IgG fraction

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Biological sample


(96-well plate kit)

IgG fraction

Reduction/Alkylation of disulfide bridges

2 Hc

2 Lc Trypsin digestion

mAB peptide digest

Waters ProteinWorks eXpress digest kit + µelution or home-designed digestion process + µelution

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Choice of 1 or 2 signature peptides


Biological sample


(96-well plate kit)

IgG fraction


2 Hc


(Addition of internal standard)

mAB peptide digest


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Choice of 1 or 2 signature peptides

LC-MS/MS MRM experiments

mAB quantification using characteristic transitions of signature peptides and internal

standard (labeled signature peptides)


Biological sample


(96-well plate kit)

IgG fraction


2 Hc


(Addition of internal standard)

mAB peptide digest


Xevo G2-XS QTOF or Xevo TQ-S mass spectrometers coupled to an Acquity M-class UPLC system,

via an IonKey source (µLC).

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Antibody quantification in plasma using MS Case study : Nivolumab, an immune check-point inhibitor

Heidelberg University Hospital – Clin. Pharmacol. Pharmacolepidemiol. - ACL | 20th June 2018 | Dr. Margaux Fresnais 7

Tumor cell recognition via TCR/MHC binding Activation of the T-cell

Activation of the immune response

+

T cell

Tumor cell

TCR MHC



Inhibition of the activation of the T-cell via PD-1/PD-L1 binding (immune checkpoints)

Inhibition of the immune response

+

TCR MHC

T cell

Tumor cell

PD-1 PD-L1



Blockade of the immune checkpoint by Nivolumab = No PD-1/PD-L1 binding

T-cell activation Activation of the immune response

+

TCR MHC

T cell

Tumor cell PD-1

PD-L1

Nivolumab



In silico digestion

A S G I T F S N S G M H W V R 15 AA

A E D T A V Y Y C A T N D D Y W G Q G T L V T V S S A S T K 30 AA

F S G S G S G T D F T L T I S S L E P E D F A V Y Y C Q Q S S N W P R 34 AA

F S G S G S G T D F T L T I S S L E P E D F A V Y Y C Q Q S S N W P R T F G Q G T K 42 AA

Heavy chain Nivolumab

Light chain Nivolumab

26 in silico peptides with 0 miscleavages


4 candidates for Nivolumab signature peptides.

Theoretical peptides depending on the digestion enzyme. Blast search against Uniprot and Swissprot databases for candidates for signature peptides.



In silico digestion

Method development in buffer

Check behavior of the candidates for signature peptides during sample preparation of intact Nivolumab (immunoprecipitation, denaturation, reduction, alkylation) first in buffer, then in plasma (iKey C18, Xevo G2-XS, MSe). Check detectability by mass spectrometry of the candidates in buffer and in biological matrix. Optimize LC method for a good separation and detection of the different peptides.

A S G I T F S N S G M H W V R 15 AA

A E D T A V Y Y C A T N D D Y W G Q G T L V T V S S A S T K 30 AA

F S G S G S G T D F T L T I S S L E P E D F A V Y Y C Q Q S S N W P R 34 AA

F S G S G S G T D F T L T I S S L E P E D F A V Y Y C Q Q S S N W P R T F G Q G T K 42 AA

Heavy chain Nivolumab

Light chain Nivolumab



2 candidates for Nivolumab signature peptides.

Method development in plasma

4 candidates for Nivolumab signature peptides: ASGI, AED, FSGS short, FSGS long.



In silico digestion



Method development MS method

Synthesis of the two unlabeled candidates. MRM parameters optimization on QTOF (ToF-MRM) and TQ-S (Intellistart). Check stability of synthetic peptides. Synthesis of the two labeled peptides for internal standard candidates. MRM parameters optimization on QTOF (ToF-MRM) and TQ-S (Intellistart). Check stability of synthetic SIL-peptides.

Good results for both candidates.

Validation of SIL-ASGI only.


2 remaining candidates for Nivolumab signature peptides: ASGI and FSGS short. Good behavior of the candidates for signature peptides during optimized sample preparation and MS analyses in buffer and in plasma. Good separation and detection of the different peptides using optimized LC method.



In silico digestion



Method development MS method

Method Validation

Clinical samples


2 remaining candidates for Nivolumab signature peptides: ASGI and FSGS short. Good behavior of the candidates for signature peptides during optimized sample preparation and MS analyses in buffer and in plasma. Good separation and detection of the different peptides using optimized LC method.

Final signature peptide: ASGI peptide and use of synthetic SIL-ASGI as internal standard. Targeted quantification using optimized MRM methods.

Last result: 0,5 µg/mL LLOQ, using TQ-S mass spectrometer.

Antibody quantification in plasma using MS For what purpose ?


How can we monitor the efficacy of an antibody-based treatment?

Isolation and analysis of the bound fraction to quantify the amount of mAB that reached the target (T-cells): pharmacodynamic study.

Quantification of free mAB in plasma for pharmacokinetic study.

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Quantification of the cell-bound fraction by MS Case study : Nivolumab, an immune check-point inhibitor


Cell-bound fraction analysis: Isolation and cell lysis

Cell lysis Conditions adapted to immunopurification

Target cell isolation

Buffy coat and red blood cells

T-cells

Complete lysis of the cell Free proteins in lysis buffer

Washing fraction

Target fraction




Immunopurification of NVL and PD-1 protein from whole cell proteins on Protein A resin

Rebuilding of immune complexes

Free proteins in coupling buffer along with immune complexes

Free PD-1 proteins and other cell proteins

Purified NVM and PD1 fraction




T-cells


Further processing

Further processing




Digestion of purified fractions and targeted quantification

Rebuilding of immune complexes



Purified NVM and PD1 fraction

Washing fraction

Target fraction




T-cells


Nivolumab cell-bound fraction and PD-1 quantification

using the surrogate peptide approach MRM or ToF-MRM analyses.





Purified NVM and PD1 fraction Immunopurification

on Protein A agarose resin

Washing fraction

Target fraction A

B

This method will help to determine the amount of Nivolumab that reached the target ( PD-1 on T-cells): Step A It can also help to quantify PD-1 proteins : Step A and B = Determination of the ratio Nivolumab/PD-1, ratio that could be linked to the efficacy of the therapy.

How are these quantification results linked to the treatment efficacy?

Summary


How to monitor the efficacy of antibody-based treatment?

Quantification of mAB fraction bound to target in the organism.

+ Pharmacokinetic study via plasma analysis.

Method currently in validation step for the quantification of two mABs: appliable to both plasma samples and bound fraction (after extended sample

preparation). So far, LOD/LLOQ of 0.5 µg/mL were reached for both

studied antibodies. Similar method can be developed for quantification of

the therapeutic target (e.g. PD-1 for Nivolumab).

Summary


How to monitor the efficacy of antibody-based treatment?

Quantification of mAB fraction bound to target in the organism.

+ Pharmacokinetic study via plasma analysis.

Method currently in validation step for the quantification of two mABs: appliable to both plasma samples and bound fraction (after extended sample

preparation). So far, LOD/LLOQ of 0.5 µg/mL were reached for both

studied antibodies. Similar method can be developed for quantification of

the therapeutic target (e.g. PD-1 for Nivolumab).

THANK YOU !

Acknowledgements


Clinical pharmacology and pharmacoepidemiology dept.

Walter-Emil Haefeli

Analytical chemistry laboratory

Jürgen Burhenne

Marius Roos-Majewski

Max Sauter

Philip Uhl

Andrea Deschlmayr

Magdalena Longo

Kevin Jansen

Kathrin Foerster

Manuela Vay

Molecular Biology Laboratory

Johanna Weiß

Dirk Theile

DKFZ and DKTK DKFZ - Division of Pediatric Neurooncology Stefan Pfister

Heidelberg DKTK coordinators Katja Engelmann Franziska Hasslinger-Pajtler

Heidelberg Immunotherapeutics Michaela Arndt

Radiopharmaceutical chemistry Walter Mier

Targeted quantification of therapeutic monoclonal ...

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