Obtaining a molecular understanding of antibody secretion

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science

Abstract

Constitutive secretion is the process by which cells deliver newly made protein to their outside. Cells can secrete a variety of different proteins and the most biologically and clinically important ones include collagen, cytokines and antibodies. Constitutive secretion is key to both normal heath and wellbeing as well as the manufacture of certain medicines such as therapeutic antibodies. Surprisingly, our understanding of this important process remains poor. This lack of basic knowledge will not only hinder improvements in the manufacturing of new drugs but also the development of novel medicines for the treatment of secretory-based diseases.

My laboratory is interested in discovering the cellular machinery required for secretion. We are identifying this machinery by comparing cells which have high levels of secretion to those which do not using a technique called mass-spectrometry which allows the abundance of proteins to be measured. Our hypothesis is that cells which are actively involved in secretion should have more of the machinery required for performing this process. The cells we are comparing for these studies are B-cells and plasma cells. Plasma cells are the antibody producing cells of the immune system and they are capable of making and secreting hundreds of millions of antibodies per day while B-cells do not secrete any antibodies. Our pilot studies have identified several new factors which we believe are involved in secretion so indicating that a more comprehensive study is warranted. Once we have identified this novel secretory machinery will then work out how these proteins function by removing them from plasma cells using a technique called RNAi and determining whether the cells can still secrete antibodies.

The knowledge generated from our research will benefit society because it will increase our understanding of a fundamental cellular process and in the future may help in the development of new manufacturing processes for therapeutic antibody production, tests for diagnosing disease and developing novel medicines for targeting diseases caused by inappropriate secretion.

Technical Summary

Constitutive secretion is the process by which newly synthesised proteins such as cytokines, extracellular matrix proteins and antibodies are exocytosed from cells. This process is key to both normal physiology and the industrial production of biological entities such as humanised antibodies. Very little is known about the post-Golgi pathways required for secretion in metazoans mainly due to the fact the molecular machinery underpinning this process has still to be elucidated. This lack of fundamental knowledge will not only hinder the industrial application of the secretory pathway but also the development of novel therapeutics for the treatment of secretory based diseases.

To address this, my group has developed a unique, physiological model of constitutive secretion based on plasma cells. Plasma cells are the antibody secreting cells of the immune system and are capable of secreting the equivalent of their own mass in antibody over a 24 hour period. To achieve this high level of secretion they have dramatically up-regulated their biosynthetic pathway in comparison to most other cell types. We propose that by profiling changes in gene expression between murine B cells and plasma cells will lead to the identification of unique factors which will provide a molecular and mechanistic understanding of post-Golgi trafficking and antibody secretion. To measure the changes in gene expression we have been using a combination of transcriptomics and proteomics. These approaches have identified several novel post-Golgi factors suggesting that a more comprehensive study is warranted. Proteins identified using these methods will have their role in antibody secretion directly determined and if they are involved in antibody secretion we will identify the cellular machinery with which they coordinate and regulate using a combination of biochemical approaches.

Planned Impact

This research will contribute to life long heath and well-being of the wider community and the development of novel approaches in industrial biotechnology as it will provide a molecular and mechanistic understanding of constitutive secretion a process required for normal physiology and the industrial production of biological entities. There are three main groups in society who will benefit from our research:

Industry: 1) Our research will aid those using the secretory pathway for industrial applications such as the production of humanised antibodies (next 5 years). The fundamental knowledge generated by this study will provide the molecular framework needed for engineering and optimising the secretory pathway. 2) This research will aid those developing novel therapies for the treatment of autoimmune based diseases and disorders caused by plasma cells dysfunction. Our research on plasma cells will identify key regulatory molecules involved in antibody secretion that in the future may be used either as bio-markers or as targets for inhibiting antibody secretion (next 10-20 years).

Health care professionals: Our research should significantly increase our understanding of intracellular transport pathways and allow for the function of novel proteins to be assigned. This in turn will aid those investigating the links between genetic mutations and human disorders (next 5 years).).

Patients: The development of novel diagnostics and therapeutics should allow for treatment and possible cure of many secretory-based diseases for which no treatments are currently available (next 10-20 years).

As well as benefiting these communities, the postdoctoral researcher employed on this grant will directly benefit from being trained in a variety of cutting edge molecular cell biological techniques and gain expertise in data analysis. The transferable skill gained in the laboratory will prepare this person for a career in either academia or industry.

Publications

10 25 50
 
Description The specific objectives of this proposal are:
1) To identify novel post-Golgi machinery required for antibody secretion using plasma cell profiling. We have performed a detailed analysis of the plasma cell transcriptome and proteome. We have identified a large number of genes unregulated during antibody secretion.

2) To determine the role of novel post-Golgi factors identified in sections 1 in antibody secretion. We are currently developing the tools to perform the genetic manipulation of plasma cells.

3) To obtain a molecular understanding of how the novel post-Golgi factors function in antibody secretion. We have identified a few novel actors are currently working on characterising their biology.
Exploitation Route We are identifying genes which facilitate high levels of secretion. These genes may be useful for engineering the secretory pathway on CHO cells. To pursue this idea we have established a collaboration with Medimmune.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description BBSRC-GSK studentship
Amount £98,212 (GBP)
Funding ID BB/R505717/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 12/2022
 
Description Proof of concept funding
Amount £98,212 (GBP)
Funding ID BB/R505717/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2018 
End 01/2019
 
Description Developing a tool kit for determining the manufacturability of new therapeutics in CHO cells 
Organisation UCB Pharma
Department UCB Celltech
Country United Kingdom 
Sector Private 
PI Contribution We have established a collaboration with UCB to develop tools for determine the manufacturability of new therapeutics in CHO cells. We are directly applying the fundamental knowledge obtained from studying antibody secretion in plasma cells.
Collaborator Contribution Our partner is providing model cell lines and therapeutics to aid in the tool development.
Impact We have obtained a BBSRC funded proof of concept grant (60K).
Start Year 2017
 
Description Engineering the secretory pathway of CHO cells 
Organisation MedImmune
Country United States 
Sector Private 
PI Contribution We are identifying novel target for engineering the secretory capacity of CHO cells and developing novel tools for measuring secretion in CHO cells.
Collaborator Contribution Medimmune are jointly funding 2 PhD students to work on the above project.
Impact This part of a larger collaboration worth approximately 1 millions pounds.
Start Year 2015
 
Description Discover night 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I co-organised a cell biology demonstration. The aim of the demonstration was to educate and inspire the general public about basic science.
We used microscopes and cell models.
Year(s) Of Engagement Activity 2014
 
Description Discovery night 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I co-organised a cell biology demonstration. The aim of the demonstration was to educate/inspire people about basic research.
We used microscopes and cell models during the presentation.
Year(s) Of Engagement Activity 2014
 
Description Krebs Fest 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I co-organised a cell biology demonstration as part of the Krebs fest. The demonstration ran for approximately 3 hours and around 500 people attended the demonstration The aim of the pod was to inspire/educate individuals about fundamental cell biology. We had microscopes, cell models and biscuit decorating.
Year(s) Of Engagement Activity 2015
URL http://krebsfest.group.shef.ac.uk