Antibody Drug-Target Engagement Measurement in Tissue Using Quantitative Affinity Extraction Liquid Chromatography–Mass Spectrometry: Method Development and Qualification

Ciccimaro, Eugene; Zhang, Yongxin; Ostanin, Dmitry V.; Suchard, Suzanne J.; MacGuire, Jamus; Xiao, Qing; Dongre, Ashok; Chimalakonda, Anjaneya; Olah, Timothy; Shipkova, Petia

doi:10.1021/acs.analchem.7b00688

Cited by 15 publications

(6 citation statements)

References 41 publications

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“…In addition, further analyses applying both LC/MS and IHC on the same samples could be used to directly correlate measured PD1 and PD‐L1 levels with the two different techniques and ultimately utilize the LC/MS findings to further qualify and/or harmonize the different clinically approved IHC assays. LC/MS/MS analyses can also be used as a multiplexing assay for the measurement of not only the biomarkers but also the drugs in tumor tissues for target engagement studies 26 …”

Section: Discussionmentioning

confidence: 99%

Immunoaffinity microflow liquid chromatography/tandem mass spectrometry for the quantitation of PD1 and PD‐L1 in human tumor tissues

Zhang

Zalaznick

Sleczka

et al. 2020

Rapid Comm Mass Spectrometry

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Rationale High tumor expression of programmed cell death protein (PD1) and programmed death‐ligand 1 (PD‐L1) is thought to be associated with positive clinical outcomes after treatment with anti‐PD1 or anti‐PD‐L1 agents. Several sensitive methods based on immunohistochemistry, ligand binding assay (LBA), and liquid chromatography/mass spectrometry involving the measurement of PD1 and PD‐L1 expression have been reported. Here, we expand on the characterization of different tumor types using a highly specific, sensitive, and robust immunoaffinity liquid chromatography/tandem mass spectrometry (IA‐LC/MS/MS)‐based method for the simultaneous quantitation of PD1 and PD‐L1 in tumor tissues. Methods Human tumor tissue samples were homogenized using a Precellys Evolution homogenizer. The samples were incubated with anti‐PD1 and anti‐PD‐L1 capture polyclonal antibodies, which were bound to magnetic beads. Following enrichment, samples were digested with trypsin. A Waters iKEY HSS T3 1.8 um (150 μm × 100 mm) column with a gradient flow rate of 3 μL/min was used for chromatographic separation, and a Waters TQ‐S triple quadrupole mass spectrometer was used for detection. Selected reaction monitoring (SRM) transitions with unit resolution for precursor/product ion masses were optimized for PD1 and PD‐L1 surrogate peptides. Results The surrogate peptides LAAFPEDR for PD1 and FTVTVPK for PD‐L1 yielded the most intense SRM transitions at m/z 459.7 > 516.2 and m/z 396.2 > 543.3, respectively, and thus were selected for the quantitation of PD1 and PD‐L1. The lower limit of quantitation for PD1 and PD‐L1 was 0.062 ng/mL with an assay range up to 10 ng/mL. Using this method, human PD1 and PD‐L1 were detected and quantified from four different types of tumor tissues. The data show that PD1 expression level was highly correlated with that of PD‐L1 in all tumor tissues analyzed here. Conclusions A highly specific and sensitive immunoaffinity microflow LC/MS/MS method for the simultaneous quantification of PD1 and PD‐L1 in tumor tissues was developed and implemented. This method combines the advantage of immuno‐capture for analyte enrichment with the high specificity of detection of multiple surrogate peptides by LC/MS/MS. The quantification of PD1 and PD‐L1 co‐expression in tumor could help evaluate their role in assessing tumor type selection and patient stratification.

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Section: Discussionmentioning

confidence: 99%

Immunoaffinity microflow liquid chromatography/tandem mass spectrometry for the quantitation of PD1 and PD‐L1 in human tumor tissues

Zhang

Zalaznick

Sleczka

et al. 2020

Rapid Comm Mass Spectrometry

Self Cite

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“…Specifically for antibodies, Ciccimaro et al [108] developed a strategy using affinity extraction (AE) liquid chromatography (LC)-mass spectrometry (MS) to directly measure the amount of drug, in that case the anti-CD40 antibody target complex, in colon biopsies of cynomolgus monkeys.…”

Section: The Assessment Of Target Organ Pk and Pd Interactionsmentioning

confidence: 99%

Translational drug discovery and development with the use of tissue‐relevant biomarkers: Towards more physiological relevance and better prediction of clinical efficacy

Florian

Flechsenhar

Bartnik

et al. 2019

Experimental Dermatology

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Due to the clinical development of drugs such as secukinumab, ustekinumab and dupilumab, major changes have been achieved in the treatment of patients diagnosed with psoriasis and atopic dermatitis. In academia and the pharmaceutical industry, research is increasingly moving towards the development of bispecific antibodies and multi‐specific nanobodies, as there is a compelling need for new treatment modalities for patients suffering from autoimmune or malignant disease. The purpose of this review is to discuss aspects of translational drug development with a particular emphasis on indications such as psoriasis and atopic dermatitis. The identification of biomarkers, the assessment of target organ pharmacokinetic and pharmacodynamics interactions and a wide range of in vitro, ex vivo and in vivo models should contribute to an appropriate prediction of a biological effect in the clinical setting. As human biology may not be perfectly reflected by approaches such as skin equivalents or animal models, novel approaches such as the use of human skin and dermal microperfusion assays in healthy volunteers and patients appear both reasonable and mandatory. These models may indeed generate highly translationally relevant data that have the potential to reduce the failure rate of drugs currently undergoing clinical development.

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“…The hybrid LC–MS assay also provides the advantage of multiplexing such as simultaneously measuring two or more structurally similar biomarkers with only one antibody pull down, followed by MS detection. [ 8 ]; quantifying both the therapeutic drug and its target to evaluate target engagement using mixed beads approach [ 7 ] as well as monitoring different isoforms of the biomarkers [ 9 ]. Most importantly, since only one capture antibody is needed in the hybrid approach, the requirement for multiple antibodies and multiple binding sites in a sandwich LBA for specificity no longer exists, and therefore, the assay development could be accelerated and the protein biomarker can be evaluated as soon as a single antibody is available in the very early stage of drug development.…”

Section: Hybrid Immunoaffinity Lc–ms Approach For Quantitative Analysmentioning

confidence: 99%

Opportunities and Challenges for Hybrid Immunoaffinity LC–MS Approach for Quantitative Analysis of Protein Biomarkers

Zhao

Zeng

2018

Future Sci. OA

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Antibody Drug-Target Engagement Measurement in Tissue Using Quantitative Affinity Extraction Liquid Chromatography–Mass Spectrometry: Method Development and Qualification

Cited by 15 publications

References 41 publications

Immunoaffinity microflow liquid chromatography/tandem mass spectrometry for the quantitation of PD1 and PD‐L1 in human tumor tissues

Immunoaffinity microflow liquid chromatography/tandem mass spectrometry for the quantitation of PD1 and PD‐L1 in human tumor tissues

Translational drug discovery and development with the use of tissue‐relevant biomarkers: Towards more physiological relevance and better prediction of clinical efficacy

Opportunities and Challenges for Hybrid Immunoaffinity LC–MS Approach for Quantitative Analysis of Protein Biomarkers

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