Flow-TriCEPS

Flow TriCEPS Service and Kit

Flow-TriCEPS

for flow cytometry

Cellular responses to ligands such as peptides, proteins, pharmaceutical drugs or entire pathogens are generally mediated through interactions with proteins expressed at the cell surface. The LRC-TriCEPS kit is designed to directly identify the target proteins of your ligand on the living cells directly using near-physiological conditions.

Dualsystems now offers the pretest TriCEPS experiments as services or as a kit.

The pretest test is designed to screen different cell types and decide which cells to use later for the identification of the unknown targets of your ligand of interest in the LRC-TriCEPS main experiment.

In case functional assays are available TriCEPS coupled ligands can also be tested to see if a similar output can be achieved with a TriCEPS coupled ligand compared to a ligand that is not coupled to TriCEPS.

As first step, the TriCEPS v.2.0 molecule is coupled to the ligand of interest (peptide, protein, Antibody, ADC or other primary amine containing molecules) and to the positive control ligand (e.g. transferrin) and negative control ligand (e.g. glycine). This coupling reaction is tested with Dot blot to assess if the coupling worked.

Then, the TriCEPS coupled ligands are added to the non-oxidized cells to assess whether the ligand binds to the unknown targets at the cell surface and TriCEPS does not interfere with the ligand receptor interaction.

Flow Cytometry tests with TriCEPS coupled ligand on oxidized cells can be further performed to test cell viability and ligand target binding as it will occur in the main LRC-TriCEPS experiment.

After successful completion of all pretests, the main experiment can be planned and carried out as described in the LRC-TriCEPS user manual choosing one of the service options provided by Dualsystems Biotech AG.

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Using Flow-TriCEPS with flow cytometry

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Ligand of interest and controls are coupled to TriCEPS. Coupling of TriCEPS to the ligand can be controlled by dot blot for protein ligands. Cells expressing the unknown targets are treated with TriCEPS coupled ligand and binding is visualized using a fluorophore.

Example of a Flow-TriCEPS experiment

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Competition experiment using Flow-TriCEPS. MDA-MB-231 cells are treated with the TriCEPS coupled ligand (orange). In order to confirm that the observed binding is due to the binding of transferrin to its target on the cells, cells are treated with different amounts of unlabeled transferrin before adding TriCEPS coupled ligand. The more unlabeled transferrin is added the smaller the shift of TriCEPS-transferrin is observed since the unlabeled transferrin competes with the TriCEPS coupled transferrin for the binding to its receptor.

TriCEPS-Transferrin on MDA cells

Flow TriCEPS-Kit flyer

Ligand-receptor capture LRC-TriCEPSflyer

Download flyer

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(Figure 1) Flow-TriCEPS is coupled to transferrin (positive control), glycine (negative control) and the ligand of interest and added on MDA-MB-231 breast cancer cells. Coupling efficiency is controlled by dot blot (Figure 2). The mean fluorescent shift is compared across the different ligands. It is expected the ligand of interest gives a larger shift than glycine. If different parameters are tested (time, temperature, pH, etc.) the condition leading to the largest shift for the ligand of interest is used for the main identification experiment.

Identify the mode of action

using the TriCEPS platforms

More information

Customers Testimonials – LRC-TriCEPS Service

Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.

Technical University of Munich

Technical University of Munich – Institute of Experimental Oncology and Therapy Research

Using LRC-TriCEPS, we aimed to identify novel direct cell surface receptors of our ligand of interest.
At any time, we experienced great support of Dualsystems Biotech. They kindly helped to find optimal conditions for our purposes and provided help with any kind of question before, during and after the experiment. LRC-TriCEPS allowed us to identify novel cell surface receptors of our ligand, which we could successfully validate in different cell types and with different biochemical assays. We can fully recommend Dualsystems Biotech and are looking forward to perform further analyses using LRC-TriCEPS.

Prof. Dr. rer. nat. Achim Krüger
Institute of Experimental Oncology and Therapy Research
Klinikum rechts der Isar, Technical University of Munich

2022-07-27T14:23:02+00:00

Technical University of Munich – Institute of Experimental Oncology and Therapy Research Using LRC-TriCEPS, we aimed to identify novel direct cell surface receptors of our ligand of interest. At any time, we experienced great support of Dualsystems Biotech. They kindly helped to find optimal conditions for our purposes and provided help with any kind of question before, during and after the experiment. LRC-TriCEPS allowed us to identify novel cell surface receptors of our ligand, which we could successfully validate in different cell types and with different biochemical assays. We can fully recommend Dualsystems Biotech and are looking forward to perform further analyses using LRC-TriCEPS. Prof. Dr. rer. nat. Achim Krüger Institute of Experimental Oncology and Therapy Research Klinikum rechts der Isar, Technical University of Munich

https://www.dualsystems.com/testimonials/technical-university-of-munich/

University of Miyazaki

We appreciate the support of Dualsystems Biotech and their LRC-TriCEPS technology we were able to identify a binding protein for our insulinotropic peptide

Hideyuki Sakoda, MD, PhD
Associate professor
Department of Biological Sciences, Faculty of Medicine, University of Miyazaki, Japan.

2022-04-19T11:45:22+00:00

We appreciate the support of Dualsystems Biotech and their LRC-TriCEPS technology we were able to identify a binding protein for our insulinotropic peptide Hideyuki Sakoda, MD, PhD Associate professor Department of Biological Sciences, Faculty of Medicine, University of Miyazaki, Japan.

https://www.dualsystems.com/testimonials/university-of-miyazaki/

Lund University Diabetes Centre

We wanted to use LRC-TriCEPS to characterise the mechanism of action of our ligand of interest. Not only were several candidate targets identified but we have successfully validated two of these receptors in insulin producing beta cells. DualSystems Biotech were very helpful and involved from study design, to experiments and reporting. Even after the initial report, DualSystems Biotech have answered follow up queries and provided assistance regarding publications. I highly recommend the LRC-TriCEPS technology and DualSystems Biotech and Paul Helbling and his team to work with.

Dualsystems-Testimonial-Lund-University

Dr. Claire L. Lyons,
Associate Researcher
Unit of Medical Protein Science
Lund University Diabetes Centre
Sweden

2021-10-25T05:58:59+00:00

https://www.dualsystems.com/testimonials/lund-university-diabetes-centre/

Australian National University

“We had a challenging project where we aimed to discover the receptor of our ligand of interest in primary human cells. Dualsystems Biotech took the challenge and provided outstanding support throughout the entire process, from the experimental design to data analysis. Using their LRC-TriCEPS technology, we could identify a candidate receptor and additional downstream interacting partners that we have now validated. We look forward to new collaborations and discoveries with Dr Paul Helbling and his team”. – Prof Carola Vinuesa, ANU

The Australian National University
Co-Director, Centre for Personalised Immunology, NHMRC Centre of Research Excellence
College of Health & Medicine
The Australian National University

2021-01-11T08:16:02+00:00

“We had a challenging project where we aimed to discover the receptor of our ligand of interest in primary human cells. Dualsystems Biotech took the challenge and provided outstanding support throughout the entire process, from the experimental design to data analysis. Using their LRC-TriCEPS technology, we could identify a candidate receptor and additional downstream interacting partners that we have now validated. We look forward to new collaborations and discoveries with Dr Paul Helbling and his team”. – Prof Carola Vinuesa, ANU The Australian National University Co-Director, Centre for Personalised Immunology, NHMRC Centre of Research Excellence College of Health & Medicine The Australian National University

https://www.dualsystems.com/testimonials/australian-national-university/

Harvard Medical School, Brigham and Women’s Hospital

We used DualSystems to help identify a potential novel receptor for a ligand that despite being studied for decades had unclear catabolism mechanisms involving its receptor mediated uptake. The DualSystems team will walk you through the process, including early validation of your ligand being used for this technology, data collection and analysis, and provide an easy to follow report on the findings. For anyone interested in receptor-ligand interactions, DualSystems and the TriCEPS approach is worth strongly considering involving in your research program, as it can quickly help identify receptors that would have been very difficult to do so without.

Maximillian Rogers, PhD
Research Scientist
Harvard Medical School, Brigham and Women's Hospital
Department of Medicine, Cardiovascular Division
Boston, MA

2020-11-09T13:27:52+00:00

https://www.dualsystems.com/testimonials/harvard-medical-school-brigham-and-womens-hospital/

Center for Biomolecular & Cellular Structure, Institute for Basic Science

“It was my great pleasure to collaborate with Dualsystems Biotech. We have been tested various proteomic analysis to identify the specific receptor for our secreted ligand. But, only LRC-TriCEPS experiment successfully gave us a right answer, because this receptor and ligand interactions were indeed relatively weaker than other general receptor and ligand interactions. The team very kindly consulted the whole experimental steps from the experimental design to the final data analysis. We are now going to use TriCEPS again for several other secreted ligands with a great hope for discovery of new receptors. Best Regards, Ho Min Kim”

Associate Professor
Graduate School of Medical Science and Engineering, KAIST
Chief Investigator
Center for Biomolecular & Cellular Structure, Institute for Basic Science (IBS)

2020-08-12T13:02:47+00:00

https://www.dualsystems.com/testimonials/center-for-biomolecular-cellular-structure-institute-for-basic-science/

Department of Internal Medicine Erasmus MC

We were fortunate to partner with Dualsystems Biotech AG to discover cell-surface targets for the short-peptide ligand we had been working on for several years. We decided to try the LRC-TriCEPS approach to identify cell-surface binding partners for the ligand after several earlier aborted attempts using different techniques. Using LRC-TriCEPS we have identified targets that we have now been able to verify functionally and biochemically using other methods. It was a pleasure to work with Dr Paul Helbling and his team who were extremely helpful guiding us through the experimental process and assisting with analysis of the data. We were impressed by the quality of the data and the ease with which we were able to run the study.

Dualsystems_testimonial_erasmus_MC

Dr Patric Delhanty
Laboratory of Metabolism and Reproduction
Department of Internal Medicine
Erasmus MC
Rotterdam, The Netherlands

2020-04-28T11:54:52+00:00

https://www.dualsystems.com/testimonials/department-of-internal-medicine-erasmus-mc/

Seoul National University

"I have used many binding assays and was in the verge of giving up. Then I have contacted Dr. Paul Helbling and his group. The LRC-TriCEPS experiment was effective and let us go over the obstacle we had during the project. We look forward to using TriCEPS in the future".

Dualsystems_Biotech_Testimonials_Seoul_National_University

Chung Hwan Cho, Ph. D. candidate
Environmental Health Microbiology Laboratory
Department of Environmental Public Health
Seoul National University

2020-01-29T14:05:52+00:00

"I have used many binding assays and was in the verge of giving up. Then I have contacted Dr. Paul Helbling and his group. The LRC-TriCEPS experiment was effective and let us go over the obstacle we had during the project. We look forward to using TriCEPS in the future". Chung Hwan Cho, Ph. D. candidate Environmental Health Microbiology Laboratory Department of Environmental Public Health Seoul National University

https://www.dualsystems.com/testimonials/seoul-national-university/

Immuno-Oncology Discovery from Bristol-Myers Squibb published in Nature

„Vista is an acidic pH selective ligand for PSGL-1“

Identification of a new immune-oncology drug target using the LRC-TriCEPS platform on primary human T-cells.

2019-10-30T06:05:00+00:00

„Vista is an acidic pH selective ligand for PSGL-1“ Identification of a new immune-oncology drug target using the LRC-TriCEPS platform on primary human T-cells.

https://www.dualsystems.com/testimonials/immuno-oncology-discovery-from-bristol-myers-squibb-published-in-nature/

The University of Oklahoma – Health Sciences Center

I was very pleased with my collaboration with the team at Dualsystems. We had to work with difficult cells and the team helped us develop modifications to the original protocol to be able to use the TriCEPS approach with our cells. The whole team was very responsive, involved, and knowledgeable and this led to a successful identification of a receptor for our ligand of interest. We have since then confirmed the interaction and we are now getting ready to publish this study, which will have implications in the treatment of ocular injuries.

Anne Kasus-Jacobi, PhD
Associate Professor of Research
University of Oklahoma Health Sciences Center
Department of Pharmaceutical Sciences
Oklahoma City, Oklahoma, USA

2019-07-02T04:38:05+00:00

https://www.dualsystems.com/testimonials/the-university-of-oklahoma-health-sciences-center/

CuroNZ Ltd

The team from DualSystems Biotech AG has been extremely helpful in delivering scientific advice for CuroNZ’s target identification journey. The quick turnaround time and the precision of NRP2945’s target identification in regard to the utilization of their TRICEPS technology impressed us very much. Meanwhile we could identify another NRP2945 receptor target that was identified by DualSystems as being recruited to the NRP2945-activated membrane receptor complex. An absolute pleasure to work with Paul Helbling’s team.

Frank Sieg, PhD
CSO
CuroNZ Ltd
Mangawhai in New Zealand

2019-02-20T12:18:04+00:00

https://www.dualsystems.com/testimonials/curonz-ltd/

University of Pittsburgh

It was a pleasure working with Paul Helbling and his team. I was impressed by their attention to detail, and optimization of all cell conditions to produce the most reliable possible results. They kept me informed at every stage of the process and shared data as soon as they were able to. Using their LRC-TriCEPS platform, we were able to follow leading candidates as binding partners for our cardiac targeting peptide and identified the most likely binding partner.

Maliha Zahid, M.D., Ph.D.
Assistant Professor
Departement of Developmental Biology
University of Pittsburgh

2018-11-15T19:35:14+00:00

https://www.dualsystems.com/testimonials/university-of-pittsburgh/

University of Oklahoma Health Sciences Center

I was very pleased with my collaboration with the team at Dualsystems. We had to work with difficult cells and the team helped our lab develop modifications to the original protocol to be able to use the TriCEPS approach with our cells. The whole team was very responsive, involved, and knowledgeable and this led to a successful identification of a receptor for our ligand of interest. We have since then confirmed this interaction and we are now getting ready to publish this study, which will have implications in the treatment of ocular injuries.

Anne Kasus-Jacobi, PhD
Assistant Professor of Research
University of Oklahoma Health Sciences Center
Department of Pharmaceutical Sciences
Oklahoma City, Oklahoma, USA

2018-04-23T11:45:16+00:00

https://www.dualsystems.com/testimonials/lrc-triceps/

Biomedical Research Institute

PUBLICATION: Li Z, Zeppa JJ, Hancock MA, McCormick JK, Doherty TM, Hendy GN, and Madrenas J. Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells. J Immunol. 2018; 200: 1471-1479.

The identification of a T cell co-receptor for staphylococcal superantigens had been challenging due to the structural features of the interaction and its kinetics. However, working with Dualstystems Biotech AG, and with Dr. Paul Helbling in particular, and using the LRC-TriCEPS technology, we were able to identify a candidate that was subsequently corroborated by biochemical and functional assays. We are very happy with this collaboration , and sincerely recommend it for the identification of novel receptor or co-receptor candidates.(Quim) Madrenas, MD, PhD, FCAHS
Chief Scientific Officer
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
Torrance, USA

2018-04-19T11:18:18+00:00

PUBLICATION: Li Z, Zeppa JJ, Hancock MA, McCormick JK, Doherty TM, Hendy GN, and Madrenas J. Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells. J Immunol. 2018; 200: 1471-1479. The identification of a T cell co-receptor for staphylococcal superantigens had been challenging due to the structural features of the interaction and its kinetics. However, working with Dualstystems Biotech AG, and with Dr. Paul Helbling in particular, and using the LRC-TriCEPS technology, we were able to identify a candidate that was subsequently corroborated by biochemical and functional assays. We are very happy with this collaboration , and sincerely recommend it for the identification of novel receptor or co-receptor candidates.(Quim) Madrenas, MD, PhD, FCAHS Chief Scientific Officer Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center Torrance, USA

https://www.dualsystems.com/testimonials/lrc-triceps-service-8/

QIMR Berghofer Medical Research Institute

“With the help of the team at Dualsystems we confirmed a potential receptor which has lead to a Nature paper submission and successful funding of a research grant. The LRC-TriCEPS experiment was straightforward and the process was cost effective. We look forward to using TriCEPS in the future.”

Anita Burgess | PhD
Hepatic Fibrosis Group
QIMR Berghofer Medical Research Institute, Australia

2017-12-12T13:21:33+00:00

https://www.dualsystems.com/testimonials/lrc-triceps-service-7/

University of Miami, Miller School of Medicine

We are very pleased with the experience and interaction on very high professional level with Dualsystems Biotech. As a result of such collaboration the new receptor MUC5B was identified in human chondrosarcoma cells for the first time for antiproliferative neuropeptide PRP-1. Read more
I would like to thank once again the company and, particularly, Dr Helbling for his attention and collaboration.

Dr Karina Galoian
Research associate professor
University of Miami, Miller School of Medicine
Department of Orthopedic surgery
Miami, Florida, USA

2017-11-16T08:07:04+00:00

We are very pleased with the experience and interaction on very high professional level with Dualsystems Biotech. As a result of such collaboration the new receptor MUC5B was identified in human chondrosarcoma cells for the first time for antiproliferative neuropeptide PRP-1. Read more I would like to thank once again the company and, particularly, Dr Helbling for his attention and collaboration. Dr Karina Galoian Research associate professor University of Miami, Miller School of Medicine Department of Orthopedic surgery Miami, Florida, USA

https://www.dualsystems.com/testimonials/lrc-triceps-service-6/

Münster University Hospital (UKM)

Dr. rer. nat. Martin Herold
Working group from Prof. Dr. med. Luisa Klotz
Münster University Hospital (UKM), Germany

2017-08-24T14:48:20+00:00

"Dualsystems Biotech’s approach to receptor identification with the TriCEPS reagent gave us a powerful platform with great support for a successful and fast data generation, when conventional approaches had previously failed."Dr. rer. nat. Martin Herold Working group from Prof. Dr. med. Luisa Klotz Münster University Hospital (UKM), Germany

https://www.dualsystems.com/testimonials/lrc-triceps-service-5/

University of Manitoba

Sari S. Hannila, PhD
Associate Professor, Department of Human Anatomy and Cell Science
Associate Member, Spinal Cord Research Centre
Max Rady College of Medicine, Rady Faculty of Health Sciences
University of Manitoba

2017-06-12T10:05:43+00:00

"Every aspect of our experience with Dualsystems Biotech was outstanding. They provided excellent customer service and technical support throughout the entire process. Their LRC-TriCEPS technology allowed us to identify several new candidate receptors for our protein of interest in rat primary neurons, and this has created new and exciting avenues for our research." Sari S. Hannila, PhD Associate Professor, Department of Human Anatomy and Cell Science Associate Member, Spinal Cord Research Centre Max Rady College of Medicine, Rady Faculty of Health Sciences University of Manitoba

https://www.dualsystems.com/testimonials/lrc-service/

The Rockefeller University

"We are very pleased with the work product of Dualsystems Biotech. The TriCEPS reagent allowed us to identify a novel ligand-extracellular matrix protein receptor interaction, which was not possible using traditional techniques. The Dualsystems team were very helpful in tailoring the experimental conditions to fit our biological question."

Manish Ponda, M.D., M.S.
Assistant Professor of Clinical Investigation
The Rockefeller University

2016-10-10T14:21:51+00:00

https://www.dualsystems.com/testimonials/lrc-triceps-service-3/

Medizinische Hochschule Hannover

“With the support of Dualsystems Biotech and their LRC-TriCEPS technology we were able to identify receptor candidates for our peptide with renoprotective properties in kidney injury.”

Dr. rer. nat. Inga Sörensen-Zender
Postdoc
Medizinische Hochschule Hannover

2016-09-26T10:26:51+00:00

“With the support of Dualsystems Biotech and their LRC-TriCEPS technology we were able to identify receptor candidates for our peptide with renoprotective properties in kidney injury.” Dr. rer. nat. Inga Sörensen-Zender Postdoc Medizinische Hochschule Hannover

https://www.dualsystems.com/testimonials/lrc-triceps-service-2/

East Tennessee State University

“For the ligand sample two unique receptors were identified. Flow cytometry analysis with siRNA induced knockdown of these proteins confirmed that the presence of the protein is needed for CTRP3 binding to occur. CONCLUSION The LRC-TriCEPS methodology was successful in identifying the receptor for CTRP3.”

Jonathan M Peterson
Assistant Professor
East Tennessee State University

2016-05-30T15:20:03+00:00

“For the ligand sample two unique receptors were identified. Flow cytometry analysis with siRNA induced knockdown of these proteins confirmed that the presence of the protein is needed for CTRP3 binding to occur. CONCLUSION The LRC-TriCEPS methodology was successful in identifying the receptor for CTRP3.”Jonathan M Peterson Assistant Professor East Tennessee State University

https://www.dualsystems.com/testimonials/lrc-triceps-service/

Igenica Biotherapeutics

«Leveraging Dualsystems Biotech novel linker technology (LRC-TriCEPS) and its analytical data processing capabilities we were able to identify the heterophilic receptor for a highly-pursued immuno-oncology target.»Dr. Edward van der Horst,
Senior Director, Preclinical Development
Igenica Biotherapeutics

2016-05-09T11:13:08+00:00

«Leveraging Dualsystems Biotech novel linker technology (LRC-TriCEPS) and its analytical data processing capabilities we were able to identify the heterophilic receptor for a highly-pursued immuno-oncology target.»Dr. Edward van der Horst, Senior Director, Preclinical Development Igenica Biotherapeutics

https://www.dualsystems.com/testimonials/lrc-triceps-service-4/

Centro de Estudos de Doenças Crónicas

New publication in Nature Communications using the LRC-TriCEPS technology in collaboration with Alisson Gontijo
« The fruitful collaboration with Dualsystems Biotech using the LRC-TriCEPS (CaptiRec) technology showed that even on insect cells receptors could be identified »

Alisson M. Gontijo,
Principal Investigator at CEDOC
Centro de Estudos de Doenças Crónicas

2015-02-24T07:32:40+00:00

New publication in Nature Communications using the LRC-TriCEPS technology in collaboration with Alisson Gontijo « The fruitful collaboration with Dualsystems Biotech using the LRC-TriCEPS (CaptiRec) technology showed that even on insect cells receptors could be identified » Alisson M. Gontijo, Principal Investigator at CEDOC Centro de Estudos de Doenças Crónicas

https://www.dualsystems.com/testimonials/captirec-service-3/

Washington University School of Medicine

"Thanks to LRC-TriCEPS (CaptiRec) technology we were able to identify a long sought receptor of our ligand using genetically-engineered cell lines with the support of Dualsystems".Fumihiko Urano, MD, PhD
Samuel E. Schechter Professor of Medicine
Washington University School of Medicine

2015-02-17T14:30:12+00:00

"Thanks to LRC-TriCEPS (CaptiRec) technology we were able to identify a long sought receptor of our ligand using genetically-engineered cell lines with the support of Dualsystems".Fumihiko Urano, MD, PhD Samuel E. Schechter Professor of Medicine Washington University School of Medicine

https://www.dualsystems.com/testimonials/captirec-service-2/

University of California San Francisco

De'Broski R. Herbert Ph.D.
Assistant Professor in Residence
University of California San Francisco (UCSF)

2015-02-17T14:27:53+00:00

"We have been trying to identify a receptor that has been sought after for nearly 30 years by laboratories across the globe. Thanks to Dualsystems service CaptiRec (LRC-TriCEPS), we now have a strong candidate receptor." De'Broski R. Herbert Ph.D. Assistant Professor in Residence University of California San Francisco (UCSF)

https://www.dualsystems.com/testimonials/captirec-service/

LRC-TriCEPS customers worldwide

Over 200 satisfied customers from 28 countries.

Customers worldwide

LRC-TriCEPS publications worldwide

Publications

LRC-TriCEPS / HATRIC-LRC Publications

Concerning the LRC-TriCEPS or HATRIC-LRC platforms.

Sensei Biotherapeutics Presents Preclinical Data at the 37th Society for Immunotherapy of Cancer (SITC) Annual Meeting

Abstract

Author

Identification of key extracellular binding proteins

Intro

Identification of key extracellular binding proteins implicate role in inflammation and fibrosis for alanyl- and aspartyl-tRNA synthetases

Abstract

Author

Akkermansia muciniphila secretes a gluc.-like peptide-1-inducing protein that improves glucose homeostasis and ameliorates metabolic disease in mice

Intro

Nature Microbiol (2021) https://doi.org/10.1038/s41564-021-00880-5

Abstract

Here, we report that A. muciniphila increases thermogenesis and gluc.-like peptide-1 (GLP-1) secretion in high-fat-diet (HFD)-induced C57BL/6J mice by induction of uncoupling protein 1 in brown adipose tissue and systemic GLP-1 secretion. We apply fast protein liquid chromatography and liquid chromatography coupled to mass spectrophotometry analysis to identify an 84 kDa protein, named P9, that is secreted by A. muciniphila. Using L cells and mice fed on an HFD, we show that purified P9 alone is sufficient to induce GLP-1 secretion and brown adipose tissue thermogenesis. Using ligand–receptor capture analysis, we find that P9 interacts with intercellular adhesion molecule 2 (ICAM-2). Interleukin-6 deficiency abrogates the effects of P9 in glucose homeostasis and downregulates ICAM-2 expression. Our results show that the interactions between P9 and ICAM-2 could be targeted by therapeutics for metabolic diseases.

Author

Neuroplastin Modulates Anti-inflammatory Effects of MANF

Intro

iScience Volume 23, ISSUE 12, 101810, December 18, 2020, DOI:https://doi.org/10.1016/j.isci.2020.101810

Abstract

Identification of Neuroplastin (NPTN) as receptor of MANF polypeptide by LRC-TriCEPS. Endoplasmic reticulum (ER) stress is known to induce pro-inflammatory response and ultimately leads to cell death. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER-localized protein whose expression and secretion is induced by ER stress and a crucial survival factor. However, the underlying mechanism of how MANF exerts its cytoprotective activity remains unclear due to the lack of knowledge of its receptor. Here we show that Neuroplastin (NPTN) is such a receptor for MANF. Biochemical analysis shows the physiological interaction between MANF and NPTN on the cell surface. Binding of MANF to NPTN mitigates the inflammatory response and apoptosis via suppression of NF-kβ signaling. Our results demonstrate that NPTN is a cell surface receptor for MANF, which modulates inflammatory responses and cell death, and that the MANF-NPTN survival signaling described here provides potential therapeutic targets for the treatment of ER stress-related disorders, including diabetes mellitus, neurodegeneration, retinal degeneration, and Wolfram syndrome.

Author

NKG2A/CD94 Is a New Immune Receptor for HLA-G and Distinguishes Amino Acid Differences in the HLA-G Heavy Chain

Intro

International Journal of Molecular Sciences. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352787/

Abstract

The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to TriCEPS and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G.

Author

Acidity changes immunology: a new VISTA pathway

Intro

Nature Immunology volume 21, pages13–16 (2020)

Abstract

Using the LRC-TriCEPS platform PSGL-1 was identified as the interaction partner of the co-inhibitor Vista on activated primary T-cells as at acidic pH mimicking the acidic tumour environment
Now, Vista tumour immunotherapy can rationally move forward
The results also suggest to use also acidity as therapeutic target for tumour therapy
Now also other interactions at acidic pH for drug candidates or co-inhibitor interactions should be tested on primary T-cells. It could explain why some drug candidates are not successful in later development.

Author

Nature Article VISTA interaction with PSGL-1 identified by LRC-TriCEPS

Intro

https://www.nature.com/articles/s41586-019-1674-5 Nature volume 574, pages565–570 (2019)

Abstract

Summary We then performed ligand-based receptor capture with VISTA–Fc chimeric protein and human CD4 + Tcells at acidic pH 19 . PSGL-1 was one of few proteins that were enriched relative to controls (Fig.3a). In addition to its well-characterized role facilitating adhesion interactions between leukocytes, platelets and endothelial cells 20,21 , PSGL-1 has been identified as a negative regulator of T cell responses in contexts of chronic viral infection, cancer, and some autoimmune diseases 22–28 .

Author

Robert J. Johnston, Linhui Julie Su, Jason Pinckney, David Critton, Eric Boyer, Arathi Krishnakumar, Martin Corbett, Andrew L. Rankin, Rose Dibella, Lynne Campbell, Gaelle H. Martin, Hadia Lemar, Thomas Cayton, Richard Y.-C. Huang, Xiaodi Deng, Akbar Nayeem, Haibin Chen, Burce Ergel, Joseph M. Rizzo, Aaron P. Yamniuk, Sanjib Dutta, Justine Ngo, Andrea Olga Shorts, Radha Ramakrishnan, Alexander Kozhich, Jim Holloway, Hua Fang, Ying-Kai Wang, Zheng Yang, Kader Thiam, Ginger Rakestraw, Arvind Rajpal, Paul Sheppard, Michael Quigley, Keith S. Bahjat & Alan J. Korman

Anti-VISTA antibody that inhibits Vista function and blocks interaction with PSGL-1 and VSIG3 proteins slows tumor growth

Intro

https://www.nature.com/articles/s41598-020-71519-4 Mehta, N., Maddineni, S., Kelly, R.L. et al. An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA. Sci Rep 10, 15171 (2020). https://doi.org/10.1038/s41598-020-71519-4 PDF

Abstract

Author

Phage resistance at the cost of virulence

Intro

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlBmediated invasion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008032 October 7, 2019
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Abstract

L. monocytogenes is a Gram-positive, food-borne, intracellular pathogen that causes severe infection in susceptible individuals. Interestingly, almost all infections are caused by a subset of strains belonging to certain serovars featuring a complex glycosylation pattern on their cell surface. Using an engineered bacteriophage that specifically recognizes these modifications we selected for mutants that lost these sugars. We found that the resulting strains are severely deficient in invading host cells as we observed that a major virulence factor mediating host cell entry requires galactose decoration of the cell surface for its function. Without this galactose decoration, the strain represents a serovar not associated with disease. Altogether, we show a complex interplay between bacteriophages, bacteria, and the host, demonstrating that cellular invasiveness is dependent upon a serovar-defining structure, which also serves as a phage receptor.

Author

TFF3 interacts with LINGO2 to regulate EGFR activation for protection against colitis and gastrointestinal helminths

Intro

Nature Communications volume 10, Article number: 4408 (2019) https://doi.org/10.1038/s41467-019-12315-1

Abstract

Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. We postulated that TFF3 interacted with its receptor through low-affinity interactions reliant upon carbohydrates because TFF3 glycosylation has been shown critical for biological activity34,35, therefore U937 cells were subjected to the TRICEPSTM protocol as a biochemical screening strategy to identify glycosylated transmembrane protein(s) on the cell surface32,36. As bait, rhTFF3 was covalently linked to the TRICEPS probe and incubated with PMA-treated U937 that had been treated with sodium periodate. Cell pellets were subjected to glycosidase digestion and peptides generated using mass spectrometry. As a positive control to account for enrichment efficiency, recombinant human>INS was used as bait for the>INS receptor. Whereas the TFF3-probe induced an 8-fold enrichment of LINGO2 (LIGO2) peptide, the>INS-probe induced a 7.76-fold-enrichment in INSR peptide (Fig. 1b). No other enriched peptides in this screen met this level of enrichment or were derived from proteins that satisfied our selection criterion (e.g., extracellular domain, transmembrane region, cytoplasmic tail).Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity.

Author

Nicole Maloney Belle 1, Yingbiao Ji 1,5, Karl Herbine 1,5, Yun Wei 2, 3,5, JoonHyung Park 1,5, Kelly Zullo1, Li-Yin Hung 1,2, Sriram Srivatsa 1, Tanner Young 1, Taylor Oniskey 2, Christopher Pastore 1, Wildaliz Nieves 4, Ma Somsouk 4 & De’Broski R. Herbert 1,2 1 Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19140, USA. 2 Division of Experimental Medicine, University of California, San Francisco, San Francisco, CA 94110, USA. 3 Department of Inflammation and Oncology, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA. 4 Division of Gastroenterology at ZSFG, University of California, San Francisco, San Francisco, CA 94110, USA. 5These authors contributed equally: Yingbiao Ji, Karl Herbine, Yun Wei, JoonHyung Park. Email: debroski@vet.upenn.edu

White Paper Ligand-Receptor Identification Methodologies Details Matter

Reference

European Biopharmaceutical Review April 2019, pages 16-20 / Maria P. Pavlou and Paul Helbling / White -Paper PDF

Abstract

Author

Validation of extracellular ligand–receptor interactions by Flow‑TriCEPS

Reference

BMC Research Notes 2018 11:863 https://doi.org/10.1186/s13104-018-3974-5 Laura A. Lopez‑Garcia, Levent Demiray, Sandra Ruch‑Marder, Ann‑Katrin Hopp, Michael O. Hottiger, Paul M. Helbling and Maria P. Pavlou Received: 28 October 2018 – Accepted: 30 November 2018- Published: 5 December 2018 PDF

Abstract

The advent of ligand based receptor capture methodologies, allows the identification of unknown receptor candidates for orphan extracellular ligands. However, further target validation can be tedious, laborious and time consuming. Here, we present a methodology that provides a fast and costefficient alternative for candidate target verification on living cells for a peptide, a protein and an antibody.

Author

Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction

Refernce

Maliha Zahid, Kyle S. Feldman, Gabriel Garcia-Borrero, Timothy N. Feinstein, Nicholas Pogodzinski, Xinxiu Xu, Raymond Yurko, Michael Czachowski , Yijen L. Wu, Neale S. Mason and CeciliaW. Lo Biomolecules 2018, 8, 147; doi:10.3390/biom8040147 Received: 24 September 2018 / Accepted: 8 November 2018 / Published: 14 November 2018

Abstract

Our previous work identified a 12-amino acid peptide that targets the heart, termed cardiac targeting peptide (CTP).We now quantitatively assess the bio-distribution of CTP, show a clinical application with the imaging of the murine heart, and study its mechanisms of transduction. Bio-distribution studies of cyanine5.5-N-Hydroxysuccinimide (Cy5.5) labeled CTP were undertaken in wild-type mice. Cardiac targeting peptide was labeled with Tc. 99m (99mTc) using the chelator hydrazino-nicotinamide (HYNIC), and imaging performed using micro-single photon emission computerized tomography/computerized tomography (SPECT/CT). Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMCs) were incubated with dual-labeled CTP, and imaged using confocal microscopy. TriCEPs technology was utilized to study the mechanism of transduction. Bio-distribution studies showed peak uptake of CTP at 15 min. 99mTc-HYNIC-CTP showed heart-specific uptake. Robust transduction of beating human iPSC-derived CMCs was seen. TriCEPs experiments revealed five candidate binding partners for CTP, with Kcnh5 being felt to be the most likely candidate as it showed a trend towards being competed out by siRNA knockdown. Transduction efficiency was enhanced by increasing extracellular potassium concentration, and with Quin., a Kcnh5 inhibitor, that blocks the channel in an open position. We demonstrate that CTP transduces the normal heart as early as 15 min. 99mTc-HYNIC-CTP targets the normal murine heart with substantially improved targeting compared with 99mTc Sestamibi. Cardiac targeting peptide’s transduction ability is not species limited and has human applicability. Cardiac targeting peptide appears to utilize Kcnh5 to gain cell entry, a phenomenon that is affected by pre-treatment with Quin. and changes in potassium levels.

Author

Maliha Zahid 1,*, Kyle S. Feldman 1, Gabriel Garcia-Borrero 1, Timothy N. Feinstein 1, Nicholas Pogodzinski 1, Xinxiu Xu 1, Raymond Yurko 2, Michael Czachowski 3, Yijen L. Wu 1, Neale S. Mason 3 and CeciliaW. Lo 1 1 Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15201, USA; ksf23@pitt.edu (K.S.F.); gag44@pitt.edu (G.G.-B.); tnf8@pitt.edu (T.N.F.); nrp30@pitt.edu (N.P.);xux@pitt.edu (X.X.); yijenwu@pitt.edu (Y.L.W.); cel36@pitt.edu (C.W.L.) 2 Peptide Synthesis Facility, University of Pittsburgh, Pittsburgh, PA 15201, USA; yurko@pitt.edu 3 Department of Radiology, University of Pittsburgh, Pittsburgh, PA 15201, USA; michael.czachowski@chp.edu (M.C.); masonns@upmc.edu (N.S.M.)

Leukocyte differentiation by histidine-rich glycoprotein/stanniocalcin-2 complex regulates murine glioma growth through modulation of anti-tumor immunity

Reference

Francis P Roche, Ilkka Pietilä, Hiroshi Kaito, Elisabet O Sjöström, Nadine Sobotzki, Oriol Noguer, Tor Persson Skare, Magnus Essand, Bernd Wollscheid, Michael Welsh and Lena Claesson-Welsh DOI: 10.1158/1535-7163.MCT-18-0097 Received January 27, 2018, Revision received April 21, 2018, Accepted June 19, 2018, Copyright ©2018, American Association for Cancer Research. PDF

Abstract

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and anti-tumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and co-localization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG treated gliomas displayed decreased numbers of Interleukin-35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control anti-tumor immunity.

Author

Francis P Roche1, Ilkka Pietilä2, Hiroshi Kaito3, Elisabet O Sjöström1, Nadine Sobotzki4, Oriol Noguer1, Tor Persson Skare1, Magnus Essand1, Bernd Wollscheid5, Michael Welsh2, and Lena Claesson-Welsh1,*

¹Department of Immunology, Uppsala University

²Department of Medical Cell Biology, Uppsala University

³Department of Immunology, Uppsala university

⁴Department of Health Sciences, ETH Zurich

⁵Department of Health Sciences, ETH Zürich

↵* Corresponding Author:

Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions

Reference

Lisa Lasswitz, Naresh Chandra, Niklas Arnberg, Gisa Gerold jmb Journal of Molecular Biology, doi.org/10.1016/j.jmb.2018.04.039 Received 15 February 2018, Revised 24 April 2018, Accepted 30 April 2018, Available online 7 May 2018.

Abstract

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.

Author

Lisa Lasswitz¹Naresh Chandra^2,3Niklas Arnberg^2,3Gisa Gerold^1,2,4
¹Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, 30625 Hannover, Germany

²Department of Clinical Microbiology, Virology, Umeå University, SE-90185 Umeå, Sweden

³Molecular Infection Medicine Sweden (MIMS), Umeå University, SE-90185 Umea, Sweden

⁴Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, SE-90185 Umea, Sweden

HATRIC-based identification of receptors for orphan ligands

Reference

Nadine Sobotzki, Michael A. Schafroth, Alina Rudnicka, Anika Koetemann, Florian Marty, Sandra Goetze, Yohei Yamauchi, Erick M. Carreira & Bernd Wollscheid Nature Communications, volume 9, Article number: 1519 (2018) doi:10.1038/s41467-018-03936-z Published online: 17 April 2018

Abstract

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu.

Author

Staphylococcal Superantigens Use LAMA2 as a Coreceptor GPCT signaling To Activate T Cells

Reference

Zhigang Li, Joseph J. Zeppa, Mark A. Hancock, John K. McCormick, Terence M. Doherty, Geoffrey N. Hendy and Joaquín Madrenas J Immunol January 15, 2018, ji1701212; DOI: https://doi.org/10.4049/jimmunol.1701212 (Published online February 5, 2018) This work was supported by the Canadian Institutes for Health Research. J.M. holds a tier I Canada Research Chair in Human Immunology. The Department of Microbiology and Immunology Flow Cytometry and Cell Sorting Facility and McGill Surface Plasmon Resonance–Mass Spectrometry Facility are supported by the Canada Foundation for Innovation.

Abstract

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein–coupled receptor (GPCR) leading to PLCβ activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.

Author

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

Reference

International Journal of Oncology, published online on: November 9, 2017 doi.org/10.3892/ijo.2017.4199 Authors: Karina Galoian, Silva Abrahamyan, Gor Chailyan, Amir Qureshi, Parthik Patel, Gil Metser, Alexandra Moran, Inesa Sahakyan, Narine Tumasyan, Albert Lee, Tigran Davtyan, Samvel Chailyan and Armen Galoyan Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed.

Abstract

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

Author

Phenotypic screening—the fast track to novel antibody discovery

Reference

ScienceDirekt, doi.org/10.1016/j.ddtec.2017.03.004

Ralph R. Minter,
Alan M. Sandercock,
Steven J. Rust^,

Department of Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge CB21 6GH, UK Available online 25 April 2017

Abstract

The majority of antibody therapeutics have been isolated from target-led drug discovery, where many years of target research preceded drug program initiation. However, as the search for validated targets becomes more challenging and target space becomes increasingly competitive, alternative strategies, such as phenotypic drug discovery, are gaining favour. This review highlights successful examples of antibody phenotypic screens that have led to clinical drug candidates. We also review the requirements for performing an effective antibody phenotypic screen, including antibody enrichment and target identification strategies. Finally, the future impact of phenotypic drug discovery on antibody drug pipelines will be discussed.

Author

Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology

Reference

PLoS One. 2016 Oct 11;11(10):e0164593. doi: 10.1371/journal.pone.0164593. eCollection 2016. Li Y¹, Ozment T², Wright GL¹, Peterson JM^1,³. We used Ligand-receptor glycocapture technology with TriCEPS-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line.

Abstract

Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the>INS receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells. Conclusion The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. Relevance The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

Author

Li Y¹, Ozment T², Wright GL¹, Peterson JM^1,³.

¹Quillen College of Medicine, Department of Biomedical Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.
²Quillen College of Medicine, Department of Internal Medicine, East Tennessee State University, Johnson City, Tennessee, United States of America.
³College of Public Health, Department of Health Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.

Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5

Reference

Manish P. Ponda and Jan L. Breslow PNAS November 8, 2016. 113 (45) E7059-E7068; published ahead of print October 24, 2016. Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan)

Abstract

MChemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.

Author

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization

Reference

Contributed by Roger Y. Tsien, August 3, 2016 (sent for review November 16, 2015; reviewed by Joshua E. Elias and Jeff W. Lichtman) Heather L. Glasgow^a,¹, Michael A. Whitney^a, Larry A. Gross^b, Beth Friedman^a, Stephen R. Adams^a, Jessica L. Crisp^b, Timon Hussain^c, Andreas P. Frei^d, Karel Novy^d, Bernd Wollscheid^d, Quyen T. Nguyen^c, and Roger Y. Tsien^a,^b,^e,²

Abstract

Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41’s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue–NP41-bound nerves, followed by hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10⁻⁷) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve “ghosts” months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.

Author

Heather L. Glasgow ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Michael A. Whitney ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Larry A. Gross ^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093; Beth Friedman ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Stephen R. Adams ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Jessica L. Crisp ^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093; Timon Hussain ^cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093; Andreas P. Frei ^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland; Karel Novy ^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland; Bernd Wollscheid ^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland; Quyen T. Nguyen ^cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093; Roger Y. Tsien ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; ^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093; ^eDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093

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