ProImmune and Pirbright collaborate to advance animal health
A strategic partnership between The Pirbright Institute and ProImmune aims to develop target-binding reagents that significantly accelerate research in animal health. Ankyrons™ are small, recombinant, target binding ankyrin repeat proteins developed by ProImmune. They are directly selected in vitro from a trillion-clone library in ribosome display and, like antibodies, can bind with high affinity to almost any target. Due to their superior properties compared to antibodies, Ankyrons™ open new avenues for scientists to study cell behaviour, offering an alternative to traditional research antibodies. The high specificity, sensitivity and reproducibility of Ankyrons™ means they can be broadly applied in biomedical science and can be targeted against all structural proteins as a cost-effective alternative to generating custom antibodies. In this collaboration, ProImmune is aiming to establish new Ankyrons™ for a range of crucial veterinary research applications where antibodies were previously unavailable. ProImmune has already generated and provided new Ankyrons™ to Pirbright for further study and validation, including for a broad range of bovine, porcine, avian and mosquito protein targets, as well as targets for significant animal diseases including Foot and Mouth Disease and African Swine Fever – all areas that have been underserved by traditional antibody technology. Pirbright’s Director of Research, Professor John Hammond, said: “The collaboration brings mutual benefits to address global animal health challenges. Multiple research groups across the Institute have worked with ProImmune to develop novel Ankyrons™ that address targets from across the tree of life including mammals, insects and viral proteins.” Dr Linda Tan, Chief Scientific Officer, ProImmune, said: “Ankyron™ technology gives researchers a better handle on biology. In this collaboration we have already demonstrated that novel Ankyrons™ can be provided rapidly to dozens of targets in areas totally unaddressed by existing research tools. We believe that over time Ankyron™ technology will have a profound impact in the wider study of biology across species and the results from this collaboration are already testament to this.” ProImmune and the Pirbright Institute hope to make significant strides in addressing global health challenges through the development of this cutting-edge research technology that will be available to researchers world-wide, studying animal physiology and disease.
Mastering Immunity Europe 2019 speakers announced and registration open
Mastering Immunity Europe 2019 will be taking place at Wolfson College, University of Oxford on 21 March. Speakers have now been announced and registration is now open.
MHC Pentamers used by team at GSK to study mRNA vaccines that can protect against several Flu strains
used by team at GSK to study mRNA vaccines that can protect against several Flu strains
Magini D. et al., PLOS One (2017) Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge http://dx.doi.org/10.1371/journal.pone.0161193
Figure:
NP-specific CD8+ T-cell responses in lungs after influenza challenge. BALB/c mice were immunized i.m. twice, 8 weeks apart, with PBS, 0.1 μg of self-amplifying mRNA (SAM®) vectors SAM(NP), SAM(M1), SAM(M1-NP), or with 0.2 μg of SAM(NP)+SAM(M1). Four weeks after the second immunization, mice were infected with PR8 virus. NP-specific CD8 T cells recruited in the lungs after the infection were characterized by flow cytometry. (a) Numbers of NP-specific CD8+ T cells determined using Pro5® MHC pentamers. Data are from individual mice (depicted as dots), while solid lanes indicate the mean±SD. (b) Cumulative frequency of Ag-specific, cytokine-secreting CD8+ T cells, indicated as absolute number per lung. The color code represents the different combinations of cytokine produced by the respective cells after in vitro stimulation with medium (m), NP147-155 peptide (NP), or M1 peptide pool (M1), as indicated. (c) Absolute number of NP-specific CD8+ T cells positive (black bar) or not (grey bar) for CD107a. Data derived from two independent and merged experiments. Statistical analyses were performed using the Mann-Whitney U test. *p<0.05; **p<0.01 compared to the PBS-treated group.
http://dx.doi.org/10.1371/journal.pone.0161193.g006
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Abbreviated abstract:
Current hemagglutinin (HA)-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to protect against mismatched circulating viruses. Inclusion of conserved proteins such as the nucleoprotein (NP) and the matrix protein 1 (M1) can increase effectiveness by eliciting cross-reactive T-cells. However, efficient priming of T-cell responses requires the right delivery system. Here we show novel self-amplifying mRNA (SAM®) vectors expressing influenza NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered with lipid nanoparticles (LNP) induce robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (TCM) and effector memory (TEM) CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP)-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. We show for the first time that the co-administration of RNA (SAM(M1-NP)) and protein (monovalent inactivated influenza vaccine (MIIV)) was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge.
Pro5® MHC Pentamer
Pro5® MHC Class I Pentamers are the market leading product for detecting antigen-specific T cells, you can count on consistent performance backed up by unrivalled customer service.
To ensure uniformity and consistency, each Pentamer is purified four times before being subjected to rigorous quality control acceptance testing.
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ProImmune launches MutaMap™ Mutational Activity Map Service
When it’s time to make final engineering decisions for your antibody or protein, MutaMap™ can help evaluate which individual point mutations to pursue. MutaMap™ is an in vitro assay system that helps explore the effect of substituting each amino acid in at each position in a protein sequence one by one with all 19 possible substitutions and find out the effect on protein activity. For example a sequence stretch of 100 amino acids will result in up to 2000 mutants to explore.
The approach of MutaMap™ is simple.
Each position of a protein of interest is mutated by site directed mutagenesis, expressed and tested for its affinity/activity. MutaMap™ does not use any surrogate measurement for affinity or activity. Cell free in vitro translation of proteins is combined with solution titration assays to measure affinity/activity. Both methods are optimized for high throughput processing of samples while still allowing for accurate measurements of affinity/activity. The technology is particularly suitable for investing the ligand binding interactions of high affinity monoclonal antibodies, down into the high femtomolar range where other approaches such as SPR struggle to deliver high throughput results.
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Mutations investigated in CDR1 and CDR3 of Avastin® heavy chain variable domain
Figure 1: Shown above is an example of a mutagenesis heatmap generated for CDR1 and CDR3 from Avastin® heavy chain variable fragment.
For binding pair interactions MutaMap™ uses high throughput solution equilibrium titration (SET) immunoassays to determine the binding affinity for each construct tested.
Figure 2: SET results for measuring the affinity of Avastin® scFv and Lucentis® scFv. The resulting titration curve is regressed according to the relevant mass action binding laws. The robustness of the SET approach ensures that this high throughput assay works well for affinities in the single digit picomolar range, as demonstrated by the tight confidence intervals.
What does MutaMap™ show you?
MutaMap™ delivers a heat map for your protein (see Figure 1 above) that shows you which point mutations lead to an increase, decrease, or no change in affinity (or other activity) or non-function of the protein when interacting with one or more of its binding partners. Effectively you can learn which mutations, one by one, are likely to be permissible or favourable in your protein in terms of the key property of binding to a ligand.
MutaMap™ therefore allows you to make informed protein engineering decisions for a range of key developability objectives which include:
Focus on position S105 in CDR-H3 of Avastin® heavy chain variable domain
Figure 3: What is clear is that the MutaMap™ heatmap shows permissible mutations, especially in CDR-H3 in cases that are not normally considered conservative, e.g. in position S105. This opens up choices for re-designing the molecule that would not normally be available based on computational assessments.
Figure 4: Position S105 in Avastin® CDR-H3 is mutated to T in in Lucentis® MutaMap™ reconfirms that this mutation is indeed beneficial for improving binding. It also shows that a number of other mutations are available to match or improve the affinity of the construct over the Avastin® wild type.
How does MutaMap™ compare to molecular evolution technologies?
Molecular evolution techniques such as phage display and other phenotype-genotype coupled randomization techniques are most commonly used in the affinity maturation process for monoclonal antibodies and other binding scaffolds. The advantage of these technologies is that they help explore a very large sequence space of combined mutations.
There comes a point however when final decisions have to be made on the implementation of a protein sequence where individual point mutations may be considered in an antibody or therapeutic protein to meet a variety of design objectives. Randomized molecular evolution is not appropriate for this step. Exploring individual point mutations is nothing new, but it has been difficult to carry this step out in very high throughput way, especially where the objective is to clone and express every mutant and then measure its affinity/activity with reasonable accuracy. This is what MutaMap™ can achieve.
Where does MutaMap™ fit in as part a project for e.g. generating a candidate monoclonal antibody for clinical development?
Figure 5:Example work flow for pre-clinial protein engineering of therapeutic monoclonal antibody; individual projects may differ.
How long does a MutaMap™ project take?
Our objective is to complete medium size projects of exploring 500-2000 mutations in approximately 8-12 weeks from receiving the customer’s protein sequence. Larger projects will take slightly longer, depending on complexity.
What is the stepwise process for carrying out MutaMap™?
First we will discuss with you the sequence space you want to explore. This may be the known paratope of a protein or the CDR and flanking regions of a monoclonal antibody.
To save time and cost you may not want to explore substituting amino acids into the sequence that are considered highly non-conservative or prone to degradation when exposed. We will also discuss what you know about the binding interaction of your protein with its target, whether you have working immunoassays for this interaction and whether the wild type protein and isolated ligand is available to work with in an immunoassay system. Depending on the nature of the interaction and the binding partners we will ensure that the base assay of binding wild type protein to target works well.
Once these details are agreed and the project is initiated we will run the mutagenesis agreed for each position, express the protein at small scale and carry out the equilibrium binding titration affinity measurement in high throughput.
What will you get?
A final technical report delivered via our secure webserver showing you the affinity or activity determination for the wild type and each mutant with confidence interval. This will be presented in various formats for ease of interpretation, including a standard heatmap.
For customers that want to carry out protein antigenicity studies in parallel, these can be carried out in approximately the same timeframe as the MutaMap™. This means that within a period of approximately 8-12 weeks we will have determined experimentally both the putative T cell epitopes and the MutaMap™ of permitted mutations in your protein sequence. This information can allow you to proceed with much better informed decisions on how to address immunogenicity related issues for your program while addressing simultaneously other developability related design decisions for your sequence.
Introducing: ProT2™ MHC Class II Tetramers
Oxford, UK, 14 January 2016
ProImmune, a leader in services for understanding and managing adaptive immune responses, announced today the introduction of ProT2™, a range of Human MHC Class II tetramer reagents for tetramer analyses in the study of antigen-specific CD4+ T cell immune responses. ProImmune’s offering represents the broadest choice of ready to order catalog and custom items in this field and will enable research into CD4+ T cell responses in a variety of disease areas including cancer, infectious diseases, allergy, autoimmunity and transplantation. Another key area for this product range is the detailed study of T cell help in unwanted immune responses to biologics.
CD4+ T cell responses are at the heart of steering the adaptive immune system directing both cytotoxic T cell responses and antibody responses mediated by B cells. ProT2™ MHC Class II Tetramers allow the direct detection and separation of single antigen-specific CD4+T cells accurately by flow cytometry.
Nikolai Schwabe, CEO of ProImmune commented: “By introducing a broad and growing ProT2™ Tetramer product range we intend to give MHC Class II tetramer analysis the emphasis it deserves in the study of CD4+ T cell responses which play such a pivotal role in adaptive immunity across so many disease and treatment modalities.”
ProT2™ Class II MHC Monomer reagents are supported by ProImmune’s expert customer service and application support, helping new users in particular to establish and optimize robust protocols in their own laboratories.
This addition to ProImmune’s class II reagent offering further complements an already extensive range of services for evaluating CD4+ T cell responses, including ProImmune REVEAL® MHC peptide binding assays, CD4+ T cell and DC T cell proliferation assays and standard immune monitoring services through ELISPOT and flow cytometry based assays.
For more information on ProT2™ reagents and their availability, or to place an order, please visit: www.proimmune.com ,email enquiries@www.proimmune.com , or call + 1 888 505 7765 in the United States or + 44 870 042 7279 in other countries.
-ENDS-
Notes to Editors:
About ProImmune – www.proimmune.com
ProImmune’s mission is to be a partner of choice for understanding and managing adaptive immune responses. It does this by offering unique solutions for preclinical and clinical immunology research, including a comprehensive and integrated antigen characterization and immunogenicity testing platform, and products and services for tracking antigen-specific immune responses with state-of-the-art ELISpot and flow cytometry techniques. ProImmune is committed to helping researcher’s achieve success through product innovation, responsive service and focused application support, saving time and money, and reducing risk.
For more information, please contact
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Jeremy Fry,
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ProImmune Introduces ProSentium™ a Unique Peptide Sequence Database to Investigate T Cell Immune System
Oxford,UK, 14 April 2015 – ProImmune, a leader in services for understanding and managing adaptive immune responses, announced today the introduction of a new antigen database service called ProSentium™, based on in vitro assay results using sequencing mass spectrometry. ProSentium data can show the precise peptide sequences from proteins of interest visible to the body’s own T cell immune system. The data collection covers many high value therapeutic targets and areas including the majority of licensed replacement factor proteins, including clotting factors, replacement enzymes, spanning development areas including oncology, cardiovascular, CNS, immunology, gastroenterology and many orphan diseases.
Under an exclusive or non-exclusive license, the bespoke database service investigates and reports peptide hits against the ProSentium database for a customer’s specific protein or protein family of interest. Results are reported as overlapping nested peptide sets and provide statistically relevant antigen sequence data that can be used to further inform drug development decisions.
Nikolai Schwabe, CEO of ProImmune commented: “ProSentium supports pivotal decision making in drug design and development with the physical evidence required for game changing approaches that target or interact with the immune system. Through our service offering, we are delighted that we can offer this ground-breaking resource to researchers so they can better understand the body’s immune response and bring new therapies to patients. ”
T cells play a critical role in all immune responses, including in fighting infections, cancer and in autoimmunity and in unwanted drug reactions. To understand T cell responses, it is crucial to understand the specific peptide antigens that the immune system recognizes and are presented on the cell surface of both normal and diseased cells. ProSentium data can be readily analyzed and reported, and is fully compatible with ProImmune’s in vitro assay services which can be combined to help investigators gain a more complete understanding of relevant immune responses at a molecular level.
For more information on ProSentium please visit: www.proimmune.com, email enquiries@www.proimmune.com , or call +1 888 505 7765 in the United States or +44 870 042 7279 inother countries.
More on ProSentium™
ProImmune’s mission is to be a partner of choice for understanding and managing immune responses. It does this by offering unique solutions for preclinical and clinical immunology research, including a comprehensive and integrated antigen characterization and immunogenicity testing platform, and products and services for tracking specific immune responses. ProImmune is committed to helping researcher’s achieve success through product innovation, responsive service and focused application support, saving time and money, and reducing risk.
Jeremy Fry
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