Understanding how aggregation influences the immune response to recombinant protein therapeutic drugs
Lead Research Organisation:
University of Manchester
Department Name: School of Biological Sciences
Abstract
Many human diseases, such as multiple sclerosis and cancer, are now treated successfully with drugs which are proteins, rather than small molecules. Protein drugs have the advantage that they are highly specific for a particular target: antibodies are a good example. They can be developed to bind with high affinity to block a specific receptor on the surface of a cell, inhibiting the progress of disease. The use of this new kind of drug in the clinic has grown rapidly over the last two decades, because protein drugs offer the potential for therapeutic intervention which would be difficult or impossible to replicate with small molecule medicines.
Although these protein drugs are generally well-tolerated and have opened up exciting new avenues for the treatment of disease, this approach has come with a specific problem. Biotherapeutic proteins (even proteins which are designed to 'look' human) can be recognised as being foreign by the immune system of the patient. This can lead to production of antibodies within the patient which bind to the protein drug and prevent it from functioning effectively. In some cases, the consequences have been more severe, leading to serious adverse health effects in patients undergoing treatment. The origin of this problem lies in the way in which the immune system recognises 'foreign' molecules: a classic question in immunology is, 'why does the immune system not attack its own host's cells?' We now know that there are sophisticated systems for eliminating or inactivating immune cells which would recognise 'self' antigens, to prevent this from happening. This phenomenon is called 'tolerance'. Many protein drugs, although apparently identical to those produced by the host, nevertheless manage somehow to breakdown tolerance and provoke an immune response. It is still unclear exactly how this happens. The aim of this proposal is to investigate this phenomenon and, ultimately, to use the information obtained to inform the design and development of more effective protein drugs in the future.
We will focus on one particular aspect which impacts on the immune response to protein drugs- a behaviour known as aggregation. Anyone who has ever boiled an egg is familiar with this behaviour: the white of the egg, after boiling, is solidified. Heat treatment pulls the structure of the protein (albumin) in the egg white apart and the individual protein chains adhere into a dense mesh. This type of behaviour is a general property of proteins, and a version of it can occur in preparations of protein drugs. The presence of aggregates in the preparation is important, as we know that they are very effective at influencing the complex cellular and molecular processes that lead to an immune response. Our proposal will investigate the different immunological responses induced by aggregates from a selection of drug-like proteins. In particular, we seek to determine how these different responses are influenced and modulated by aggregate size, which is the main criterion used to characterise them.
Although these protein drugs are generally well-tolerated and have opened up exciting new avenues for the treatment of disease, this approach has come with a specific problem. Biotherapeutic proteins (even proteins which are designed to 'look' human) can be recognised as being foreign by the immune system of the patient. This can lead to production of antibodies within the patient which bind to the protein drug and prevent it from functioning effectively. In some cases, the consequences have been more severe, leading to serious adverse health effects in patients undergoing treatment. The origin of this problem lies in the way in which the immune system recognises 'foreign' molecules: a classic question in immunology is, 'why does the immune system not attack its own host's cells?' We now know that there are sophisticated systems for eliminating or inactivating immune cells which would recognise 'self' antigens, to prevent this from happening. This phenomenon is called 'tolerance'. Many protein drugs, although apparently identical to those produced by the host, nevertheless manage somehow to breakdown tolerance and provoke an immune response. It is still unclear exactly how this happens. The aim of this proposal is to investigate this phenomenon and, ultimately, to use the information obtained to inform the design and development of more effective protein drugs in the future.
We will focus on one particular aspect which impacts on the immune response to protein drugs- a behaviour known as aggregation. Anyone who has ever boiled an egg is familiar with this behaviour: the white of the egg, after boiling, is solidified. Heat treatment pulls the structure of the protein (albumin) in the egg white apart and the individual protein chains adhere into a dense mesh. This type of behaviour is a general property of proteins, and a version of it can occur in preparations of protein drugs. The presence of aggregates in the preparation is important, as we know that they are very effective at influencing the complex cellular and molecular processes that lead to an immune response. Our proposal will investigate the different immunological responses induced by aggregates from a selection of drug-like proteins. In particular, we seek to determine how these different responses are influenced and modulated by aggregate size, which is the main criterion used to characterise them.
Technical Summary
The overall aim of the proposal is to study the quality and vigour of the immune response to selected model biotherapeutic proteins, and the aggregates generated from them. Our experimental approach will combine studies using a well characterised (BALB/c strain) mouse model of protein immunogenicity and allergenicity, cultured human and mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMC). The programme will comprise the following tasks:
Task 1: Production and characterisation of recombinant mouse and human protein aggregates for use in immunological investigations.
Task 2: To determine whether aggregation influences the stimulation of adaptive immune responses in vivo. A mouse (BALB/c) model will be used to monitor whether aggregation of recombinant mouse proteins impacts on:
a) IgG isotype distribution
b) IgE antibody production
c) cytokine production (Interleukin [IL]-2, IL-4, IL-6, IL-10, IL-13, IL-17 and IL-22)
d) the balance between antigen-driven CD4+ and CD8+ T lymphocyte responses
e) the frequency of regulatory T cells (Tregs)
f) the number and phenotype of CD11c+ dendritic cells (DC) in the spleen.
Task 3: To determine whether aggregation influences the stimulation of primary immune responses in vitro. To address this question, the elicitation of human T and B cell responses in vitro by aggregated and non-aggregated human/humanised proteins will be characterised.
Task 4: Characterisation of the mechanistic basis for differential effects of aggregated and non-aggregated proteins to cause immune stimulation. The investigation will determine whether aggregated and non-aggregated proteins display a differential potential to
a) provoke cytokine production by peripheral blood mononuclear leukocytes (PBMC).
b) drive the activation of mouse and human DC in vitro.
Task 1: Production and characterisation of recombinant mouse and human protein aggregates for use in immunological investigations.
Task 2: To determine whether aggregation influences the stimulation of adaptive immune responses in vivo. A mouse (BALB/c) model will be used to monitor whether aggregation of recombinant mouse proteins impacts on:
a) IgG isotype distribution
b) IgE antibody production
c) cytokine production (Interleukin [IL]-2, IL-4, IL-6, IL-10, IL-13, IL-17 and IL-22)
d) the balance between antigen-driven CD4+ and CD8+ T lymphocyte responses
e) the frequency of regulatory T cells (Tregs)
f) the number and phenotype of CD11c+ dendritic cells (DC) in the spleen.
Task 3: To determine whether aggregation influences the stimulation of primary immune responses in vitro. To address this question, the elicitation of human T and B cell responses in vitro by aggregated and non-aggregated human/humanised proteins will be characterised.
Task 4: Characterisation of the mechanistic basis for differential effects of aggregated and non-aggregated proteins to cause immune stimulation. The investigation will determine whether aggregated and non-aggregated proteins display a differential potential to
a) provoke cytokine production by peripheral blood mononuclear leukocytes (PBMC).
b) drive the activation of mouse and human DC in vitro.
Planned Impact
The proposed research will have beneficiaries from four distinct groupings:
1. Pharmaceutical companies developing biotherapeutic medicines and vaccines.
The relevance of the proposed research to the biopharmaceutical sector is obvious and requires little elaboration, given that Lonza are joint applicants and contributing half of the cost of this programme. Specifically, the research will enable the design of more effective therapeutics through the identification and control of features leading to immune responses; this can be applied to both biotherapeutics, where the goal is to minimise immune responses, and vaccines, where stimulation of an effective immune response is important. Results will be disseminated through publications, conference presentations and also the informal contacts which the academic applicants (JPD/IK/RJD) have with industry (subject to agreement with the industrial partner, Lonza). Lonza will also use the results to improve its service offering for the design of therapeutics. Pharmaceutical companies will be able to innovate new biotherapeutics with greater confidence if they can identify and control features (such as aggregation size populations) which are less likely to provoke immune responses.
2. Training of staff employed on the grant
The UK hosts a large and expanding bioprocessing community: there are around 15 members of the BBSRC, EPSRC and Industry-funded Bioprocessing Research Industry Club. Promoting the development of training in skills areas relating to bioprocessing products is a priority, and this proposal will contribute to that overall goal. In particular, the combination of protein biochemistry, immunology and animal experimentation will help to develop skills in the employed research staff which will promote their employability and benefit the sector as a whole. Furthermore, as is often the case, funding of a research proposal generates a 'core' of scientific expertise which acts as a nucleus for further industrial collaborations and teaching/training of Masters and PhD students.
3. Regulatory authorities
Scientists working for regulatory authorities constitute the third group of more general beneficiaries for this proposal. Examples include the US Federal Drug Administration (FDA), and, in the UK, the Medicines and Healthcare products Regulatory Agency (MHRA) and the National Institute for Biological Standards and Control (NIBSC; until recently part of the Health Protection Agency). Clearly, the problem of unwanted immunogenicity in biotherapeutics has a direct bearing on the regulation of new medicines. A substantial body of scientists work within these and similar regulatory authorities on medicine safety: research which helps to improve guidelines and the monitoring of new drugs will obviously be of value to them. Representatives from regulatory bodies are regular attendees at national and international conferences, and so will be informed of our results through that route, as well as having access through our publications and personal contacts.
4. Patients who will be treated with biotherapeutic medicines
Ultimately, the most important grouping to benefit from this proposal will be future patients. The global market for biological medicines in 2011 was $94bn (Thompson Reuters) and is continuing to grow. The numbers of people who will be treated with such drugs in the next 10 years is likely to be in the hundreds of thousands. Reduction or removal of those types of aggregates which would otherwise potentiate the immunogenicity of a drug will have obvious health benefits. An improved understanding of the immunological mechanisms underlying the response to biotherapeutics is therefore going to be an important part of the necessary scientific background which is taken into account when decisions are made about aggregate content in a biological medicine.
1. Pharmaceutical companies developing biotherapeutic medicines and vaccines.
The relevance of the proposed research to the biopharmaceutical sector is obvious and requires little elaboration, given that Lonza are joint applicants and contributing half of the cost of this programme. Specifically, the research will enable the design of more effective therapeutics through the identification and control of features leading to immune responses; this can be applied to both biotherapeutics, where the goal is to minimise immune responses, and vaccines, where stimulation of an effective immune response is important. Results will be disseminated through publications, conference presentations and also the informal contacts which the academic applicants (JPD/IK/RJD) have with industry (subject to agreement with the industrial partner, Lonza). Lonza will also use the results to improve its service offering for the design of therapeutics. Pharmaceutical companies will be able to innovate new biotherapeutics with greater confidence if they can identify and control features (such as aggregation size populations) which are less likely to provoke immune responses.
2. Training of staff employed on the grant
The UK hosts a large and expanding bioprocessing community: there are around 15 members of the BBSRC, EPSRC and Industry-funded Bioprocessing Research Industry Club. Promoting the development of training in skills areas relating to bioprocessing products is a priority, and this proposal will contribute to that overall goal. In particular, the combination of protein biochemistry, immunology and animal experimentation will help to develop skills in the employed research staff which will promote their employability and benefit the sector as a whole. Furthermore, as is often the case, funding of a research proposal generates a 'core' of scientific expertise which acts as a nucleus for further industrial collaborations and teaching/training of Masters and PhD students.
3. Regulatory authorities
Scientists working for regulatory authorities constitute the third group of more general beneficiaries for this proposal. Examples include the US Federal Drug Administration (FDA), and, in the UK, the Medicines and Healthcare products Regulatory Agency (MHRA) and the National Institute for Biological Standards and Control (NIBSC; until recently part of the Health Protection Agency). Clearly, the problem of unwanted immunogenicity in biotherapeutics has a direct bearing on the regulation of new medicines. A substantial body of scientists work within these and similar regulatory authorities on medicine safety: research which helps to improve guidelines and the monitoring of new drugs will obviously be of value to them. Representatives from regulatory bodies are regular attendees at national and international conferences, and so will be informed of our results through that route, as well as having access through our publications and personal contacts.
4. Patients who will be treated with biotherapeutic medicines
Ultimately, the most important grouping to benefit from this proposal will be future patients. The global market for biological medicines in 2011 was $94bn (Thompson Reuters) and is continuing to grow. The numbers of people who will be treated with such drugs in the next 10 years is likely to be in the hundreds of thousands. Reduction or removal of those types of aggregates which would otherwise potentiate the immunogenicity of a drug will have obvious health benefits. An improved understanding of the immunological mechanisms underlying the response to biotherapeutics is therefore going to be an important part of the necessary scientific background which is taken into account when decisions are made about aggregate content in a biological medicine.
Organisations
Publications
Austerberry J
(2021)
Use of Peptide Microarrays for Fast and Informative Profiling of Therapeutic Antibody Formulation Conditions.
in Molecular pharmaceutics
Austerberry JI
(2019)
Arginine to Lysine Mutations Increase the Aggregation Stability of a Single-Chain Variable Fragment through Unfolded-State Interactions.
in Biochemistry
Eyes TJ
(2019)
Identification of B cell epitopes enhanced by protein unfolding and aggregation.
in Molecular immunology
Ratanji KD
(2017)
Influence of Escherichia coli chaperone DnaK on protein immunogenicity.
in Immunology
| Description | Many human diseases are now treated successfully with drugs which are proteins, rather than small molecules. Examples include the successful drug Adalimumab, which is used to treat rheumatoid arthritis. Protein drugs have the advantage that they are highly specific for a particular target: they can bind with high affinity to block a specific protein, inhibiting the progress of disease. The use of this new kind of drug in the clinic has grown rapidly over the last two decades. This revolution has been made possible through the development of monoclonal antibodies, which will recognize only one particular 'target', or antigen. They allow us to produce, in principle, unlimited quantities of a specific antibody in a lab or production facility. Monoclonal antibodies have found many applications in scientific research, as well as their use in medicine. Although therapeutic monoclonal antibodies are generally well-tolerated and have opened up exciting new avenues for the treatment of disease, this approach has come with a specific problem. Biotherapeutic proteins (even proteins which are designed to 'look' human) can be recognised as being foreign by the immune system of the patient. This can lead to production of antibodies within the patient which bind to the protein drug and prevent it from functioning effectively (so-called 'anti-drug antibodies', or 'ADAs'). In some cases, the consequences can be more severe, leading to serious adverse health effects in patients undergoing treatment. More often, however, the formation of ADAs impairs the efficacy of drug therapy. The degree to which individual patients form ADAs varies considerably from person to person, although it is not routinely monitored. Therapeutic antibodies are very expensive, so prolonging treatment when it is ineffective is wasteful and expensive. A key factor in promoting ADA formation is aggregation- the formation of large, amorphous particles by antibodies in solution. We have found that we can extract more detailed information on the ADA response to aggregates by using an approach called 'immunoprofiling'. This allows us to obtain a profile of the ADAs formed. First, we showed proof or principle that this method can be used to profile ADA responses to aggregated proteins in mice. Second, we have applied the same principle to study the immunoprofiles using sera derived from human patients who are in receipt of monoclonal antibody therapy for inflammatory diseases. These observations, in turn, have led to us exploring commercial application of this technology, which could be used as a diagnostic procedure to predict disease susceptibility and likely outcomes of treatment. We have secured funding for commercial development in this area, which is ongoing. |
| Exploitation Route | The results will open up new ways in which to assay ADA formation and may ultimately help to promote more economical use of therapeutic antibodies. They may also inform ways in which new therapeutic antibodies, and related macromolecules, are developed in industry. |
| Sectors | Healthcare,Manufacturing, including Industrial Biotechology |
| Description | We have been awarded £99,000 from UMI3, the University of Manchester Innovation Company, to exploit the results arising from this grant. The application is centred on the use of antibody affinity profiling to stratify patients suffering from inflammatory conditions, to predict disease susceptibility and/or likelihood of success of treatment using anti-TNF alpha therapies. |
| First Year Of Impact | 2017 |
| Sector | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |
| Description | MRC P2D |
| Amount | £12,876 (GBP) |
| Organisation | University of Manchester |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 10/2018 |
| End | 12/2018 |
| Description | Wellcome Trust Access to Expertise |
| Amount | £17,251 (GBP) |
| Organisation | University of Manchester |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 09/2018 |
| End | 02/2019 |
| Title | Fast and informative profiling of formulation conditions using peptide microarrays |
| Description | Methods to optimize the solution behavior of therapeutic proteins are frequently time-consuming, provide limited information, and often use milligram quantities of material. Here, we present a simple, versatile method that provides valuable information to guide the identification and comparison of formulation conditions for, in principle, any biopharmaceutical drug. The subject protein is incubated with a designed synthetic peptide microarray; the extent of binding to each peptide is dependent on the solution conditions. The array is washed, and the adhesion of the subject protein is detected using a secondary antibody. We exemplify the method using a well-characterized human single-chain Fv and a selection of human monoclonal antibodies. Correlations of peptide adhesion profiles can be used to establish quantitative relationships between different solution conditions, allowing subgrouping into dendrograms. Multidimensional reduction methods, such as t-distributed stochastic neighbor embedding, can be applied to compare how different monoclonals vary in their adhesion properties under different solution conditions. Finally, we screened peptide binding profiles using a selection of monoclonal antibodies for which a range of biophysical measurements were available under specified buffer conditions. We used a neural network method to train the data against aggregation temperature, kD, percentage recovery after incubation at 25 °C, and melting temperature. The results demonstrate that peptide binding profiles can indeed be effectively trained on these indicators of protein stability and self-association in solution. The method opens up multiple possibilities for the application of machine learning methods in therapeutic protein formulation. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | We are currently in discussion with an SME to apply this method to the formulation of their biological medicines. We have also promoted the method at various meetings (eg BioProNet2) |
| URL | https://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.1c00543 |
| Title | Use of synthetic peptide arrays to differentiate antibody responses to aggregates of therapeutic proteins |
| Description | B-cell epitope profiling differentiates immunogenic responses to protein aggregates Aggregation of monoclonal antibodies and therapeutic proteins has been well documented as a key factor in the generation of undesirable immunogenicity which can lead to the shortening of serum half-life, neutralisation of function and adverse clinical consequences. Aggregation may enhance immunogenic potential through increased antigen presentation, repetitive antigen patterning and generation of non-native epitopes. Using peptide microarray technology, we have designed a model system that examines the changes in the B-cell epitope repertoire between native and aggregated proteins. Two recombinant human antibody fragments were selected as model proteins; VH (heavy V domain) and scFv (single chain of heavy and light V domains). VH and scFv proteins were aggregated by heating to 60°C and 40°C respectively. Aggregation was confirmed by dynamic light scattering. BALB/c strain mice were immunised with either protein (monomer or aggregate). Anti-VH or anti-scFv serum IgG titres were analysed by ELISA. B-cell epitopes were screened by peptide array consisting of overlapping peptide libraries for both VH and scFV proteins. Serum samples were analysed on the arrays and total IgG and IgG isotype measured by fluorescence detection. Both proteins form irreversible aggregates with particle size in the range of 1-3µm. Immunisation with aggregate protein demonstrated an increase in IgG titre compared to monomer and a significant switch to IgG2a isotype for both proteins, indicative of a T helper type 1 response. Analysis of sera by epitope array showed differential IgG and isotype signatures between aggregate and monomer immunised serum. Recognition of a small number of epitope peptides was significantly increased in aggregate immunised serum compared to monomer. It is proposed that protein aggregation changes the B cell epitope landscape and array technology can aid identification of enhanced immunogenic sequences. Knowledge of aggregate related epitopes will aid development of therapeutic proteins and the analysis of anti-drug antibodies in patients. |
| Type Of Material | Technology assay or reagent |
| Provided To Others? | No |
| Impact | Development of this method is in progress. |
| Description | BRIC dissemination event & BioProNet, Manchester 21-22 October |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster display, as a focus for general discussion and dissemination of our work to industry and academia. |
| Year(s) Of Engagement Activity | 2015 |
| Description | BioprocessUK & BioProNet, Liverpool 25-26 Nov |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster display, as a focus for general discussion and dissemination of our work to industry and academia. |
| Year(s) Of Engagement Activity | 2014 |
| Description | Faculty of Life Sciences Open Day |
| 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 | The Faculty of Life Sciences runs an annual Open Day (in May). The Derrick group ran a stall as part of this event, which was designed to illustrate and explain ongoing work in the lab. Particular effort was given to developing display objects, using 3D printing technology, which allowed children and families to explore the structures of antibodies and viruses. These activities prompted numerous questions and interest from school groups (who came through on one day) and the general public (who attended on the next day). £950 was obtained from the British Society for Immunology to support this work. |
| Year(s) Of Engagement Activity | 2015,2016 |
| Description | PEGS Europe Lisbon 2016 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Talk to present the translational potential of research work being conducted in the group. Gave rise to interactions with interested parties from industry. |
| Year(s) Of Engagement Activity | 2016 |
| Description | Protein Engineering Summit 2016, Boston |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | An invited talk at the Protein Engineering Summit 2016, Boston, USA 27 April - 1 May 2016. Also, posters were given by members of the group. Presentation of our data on aggregation of biologics stimulated discussion with many practitioners and industry representatives. Follow-up discussions and meetings have been arranged. |
| Year(s) Of Engagement Activity | 2016 |
| Description | Talk on Immunogenicity |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Talk on the relationship between the immunogenicity of biopharmaceutical drugs, their conformational state and aggregation, in an industrial context. The talk was given by the PDRA funded on the grant- a valuable career development opportunity- and precipitated further discussions with industry stakeholders. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Year 10 pupils work experience |
| 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 | Schools |
| Results and Impact | Carried out a laboratory demonstration on protein expression. Specifically targeted at pupils from local schools with an interest in Life Sciences who wouldn't otherwise have the opportunity to carry out work experience activities, with the aim of raising awareness of careers pathways and higher education. |
| Year(s) Of Engagement Activity | 2016 |