Full title: Characterisation of host immune variations that influence live-attenuated Yellow Fever vaccine adaptive immune responses. Vaccines are one of the most effective tools in controlling viral pandemics. However, adaptive immune responses to vaccines can vary widely between individuals. Since the live-attenuated Yellow Fever vaccine is known to induce robust adaptive immune responses, we investigated that baseline molecular signatures that are associated with the magnitude of humoral and cellular immune responses. We determined that the baseline signatures associated with humoral and cellular immune responses are distinct and could be influenced by both genetic and environmental factors. Our results demonstrate that host intrinsic factors can influence vaccination outcome, and suggest that changes in lifestyle and environment can be explored to improve vaccine immunogenicity. Dr. Kuan Rong Chan is a Principal Research Scientist at Duke-NUS Medical School, with close to 15 years of research experience in virology, immunology and bioinformatics. His research applies systems biology approaches to the field of virology and vaccinology, to understand how individuals respond to viral infections and vaccines. His research is currently funded by the Open Fund-Individual Research Grant from the National Medical Research Council.
Generic peptide therapeutics enable the increased access to low cost and high-quality medicines. Current regulatory guidelines allow for the filing of highly purified synthetic generic peptides based on data from in vitro immunogenicity risk assessment and on the characterization of impurity profiles as compared with the approved reference drug. Here we highlight current methods for assessing both adaptive and innate immunogenicity risks as they have been adapted to support these filings. Andrew earned his PhD in molecular biology from the University at Albany under the direction of Professor Robert Osuna, identifying and characterizing post translational modifications of the transcription factor DksA in E. coli. He joined ProImmune in 2019 after completing his doctorate and currently works on providing innovative solutions for clients that deeply improve our understanding of both desired and unwanted immune responses.
In this presentation, Dr Kei Kishimoto from Selecta BioSciences discusses their ImmTOR technology, which combines a nanoparticle with the approved anti-inflammatory and immunomodulatory drug rapamycin to induce antigen-specific immune tolerance. Dr Kei shares compelling evidence from their Phase I clinical trial that ImmTOR co-administered with the highly immunogenic gout treatment Pegadricase is able to suppress the antibody response to the treatment in a dose-dependent manner. ImmTOR is currently in Phase III clinical trials, with the current trial expected to be completed in Q1 of 2023. Dr Kishimoto also presents the next generation of the ImmTOR technology, ImmTOR-IL, which combines ImmTOR with an engineered, T-reg selective IL-2 mutein. In mice, a single dose of ImmTOR with IL-2 mutein induces massive expansion of antigen specific T-regulator cells when co-administered with the antigen of interest. Treg-selective IL-2 has considerable potential to work synergistically with the ImmTOR platform in the clinic, with ImmTOR mitigating the immunogenicity of the engineered IL-2 mutein, and the Treg-specific IL-2 enabling dose sparing of ImmTOR. Dr Kei Kishimoto is the Chief Scientific Officer of Selector Biosciences. Prior to joining Selector, he held leadership positions at Momenta, Millennium and Boehringer Ingelheim. He completed his Doctorate at Harvard and received his Postdoctoral training at Stanford.
In this talk, Dr Mike Zimmer introduces one of Moderna’s fledgling projects for their rare disease group, engineering a less immunogenic bacterial protease for treatment of IgA Nephropathy. IgA Nephropathy (Berger’s disease) is a rare autoimmune disease for which is currently poorly understood. It is characterised by deposition of IgA-containing immune complexes in the glomerulae of the kidneys and can lead to renal failure. Many gut bacteria secrete IgA proteases as part of host defence. It has been previously shown that these IgA proteases can be used in vitro to degrade with IgA1 complexes which cause IgA nephropathy. Further, IgA1 protease treatment can reverse mesangial deposits in transgenic mice expressing human IgA1 and soluble IgA Fc receptor I (sCD89) proteins. Moderna have developed modified bacterial IgA proteases which are amenable to expression in eukaryotic cells, and are active in in vivo mouse models. They are now working to tolerise or de-immunise IgAP via two approaches – Liver-mediated immune tolerance and targeted mutagenesis. Using ProImmune’s ProPresent® Antigen Presentation Assay and our ProMap® naïve T cell proliferation assay, Moderna were able to identify peptides from IgAP which are presented to the immune system by APCs, and then determine which of these cause a helper T cell immune response. They were able to identify 30 peptides which were presented on antigen presenting cells, none of which produced a helper T cell response. Having narrowed down potential epitopes to these 30 peptides, Dr Zimmer’s team were able to conduct in silico mutagenic scanning of potentially immunogenic peptides, revealing possible sites for de-immunisation. Ultimately, the team identified the amino acids which contributed to the potential immunogenicity of the peptides identified by ProImmune. They have since successfully mutated many of these amino acids by targeted mutagenesis. Their planned next step is to re-express their protein with the introduced mutation in their mouse model and determine whether the mutations allow for repeat dosing. They also plan to compare the immunogenicity of the original and mutated proteins in ProImmune’s ProScern® assay, which will measure the extent to which these proteins induce CD4+ T cell proliferation. They expect that they will ultimately develop a product which can degrade human IgA nephrotic deposits through periodic dosing.
Aro Biotherapeutics is a preclinical stage biotechnology company focused on the discovery and development of Centyrins, a new class of small, ultra-stable and highly soluble proteins engineered to specifically bind antigens with high affinity. Aro has created therapeutic lead candidates combining Centyrins specific for the transferrin receptor (CD71) with siRNAs that target Gys1, an enzyme responsible for glycogen production. CD71 Centyrin-Gys1 siRNA conjugates represent a new modality for the treatment of Pompe disease, a muscle glycogen storage disease. The CD71 Centyrin-Gys1 siRNA is highly effective at reducing Gys1 mRNA in vivo in an animal model of Pompe disease. Centyrin-drug conjugates therefore provide a means to deliver siRNAs to modulate genes once considered “undruggable”. We have shown that Centyrins enable the delivery and have extended exposure in early endosomes to facilitate delivery and endosomal release of siRNAs. Despite the long residence time in endosomes, utilizing the suite of Proimmune assays, we have shown that Centyrins have a low probability of eliciting an immune response in humans. Potential mechanisms for the low predicted immunogenicity will be discussed. Steven Nadler, PhD, is currently Senior Vice President and Head of Discovery and Translational Research at Aro Biotherapeutics. Steve has more than 30 years of experience in drug discovery, development and translational research prior to Aro Biotherapeutics. As the former Executive Director and Head of Immunoscience, Immuno-oncology and Oncology Discovery Translational Research at Bristol-Myers Squibb, he was solely responsible for establishing the company’s Immunoscience and Immuno-oncology Discovery Translational Research group. During his tenure at Bristol-Myers Squibb, he took on roles of increasing responsibility in the autoimmune and oncology therapeutic areas, where he was instrumental in the discovery and development of five biologic and small molecules which entered the clinic for autoimmune diseases. He also led early clinical development for three of these biologics. Steve was an integral member of the teams which brought both ORENCIA® and NULOJIX® from discovery to the market. Steve received his PhD from the University of Texas at Houston, followed by postdoctoral studies at Yale Medical School. He has authored more than 80 publications and is co-inventor on over 10 patents.
Liver sinusoidal endothelial cells (LSECs) are capable to induce tolerance against blood borne antigens, e.g. those originating from food. We mimic those antigens by small peptide-loaded nanoparticles which are intravenously injected. After active and rapid uptake of those nanoparticles by the LSECs, peptide-specific regulatory T cells are generated which confer tolerance against one or more specific antigens in the periphery of the body. A one-time application of our peptide-loaded nanoparticles in EAE mice led to complete tolerance against this MS-like disease. We will discuss the underlying mechanism, the extension to other diseases and applications as well as the fate of the nanoparticles in the body. Timm Jessen is biochemist and received his academic career in Kiel, Munich, Cambridge and Boston. He spent six years in pharma before he assumed the role of the CSO in Evotec between 1997 and 2004. Throughout his subsequent self-employment he founded several companies of which Topas focuses on the translation of this academic invention from the University Hospital of Hamburg into therapeutic products in the area of autoimmune diseases and allergies. Today Timm is the CEO of Topas Therapeutics.
Edmund Neo is ProImmune’s Immunology Sales Specialist located in Singapore representing the company in the Asia-Pacific Region. Edmund graduated from the National University of Singapore and worked with Abbott Laboratories before joining ProImmune in 2019. He has played a pivotal role in the company by expanding businesses in his region.
The emerging and re-emerging viruses e.g. SARS-CoV-2 and influenza viruses pose a great threaten to public health. Considering the special features such as the conserved targets and long-term durability, the host T-cell provides an unneglectable role during the anti-virus immunity. We established a T cell-based test, surveillance and research platform to investigate the mechanism by which the human T cell immunity reacts to the viruses. We have screened a series of T cell epitopes from SARS-CoV, SARS-CoV-2, MERS-CoV, influenza viruses, ZIKV, HBV, EV71, SFTSV, etc. and utilized these epitopes to investigate the antigen-specific cellular immune responses in the patients, convalescents, and vaccinated populations. In addition, we investigated the T cell immune features of different vertebrate animals by determined the 3D structures of a diverse MHC molecules, i.e. HLA (human), H-2 (mouse), Mamu (monkey), SLA (swine), BF2 (chicken), BoLA (bovine), DLA (dog), ELA (horse), RLA (rabbit), FLA (feline) and Ptal (bat), etc. The understanding of T cell mediated immune mechanism to the emerging virus infections will benefit the development of the peptide-based vaccines. Prof. William Liu is the Deputy Director of the Chinese National Influenza Center. He graduated from the School of Public Health, Peking University Health Science Center in 2005. He received his Ph.D degree from Institute of Microbiology, Chinese Academy of Sciences and received his postdoctoral training at Yale School of Medicine. The main research focus of his group is the T-cell recognition of emerging and re-emerging viruses, i.e. SARS-CoV-2, influenza viruses etc. He has published more than 120 papers in leading journals such as Nature, Immunity, PNAS and J Immunology and J Virology. He is the deputy Editor-in-Chief of Biosafety and Health, Invited Guest Editor of Frontiers in Immunology and Science China Life Sciences, and editors for Infectious Diseases & Immunity. Dr. Liu has worked as Chinese Public Health Team leader in Africa during the Ebola Epidemic. During the COVID-19 pandemic, he worked in Wuhan for over 100 days.
The team at Vaxine Pty Ltd is focussed on development of vaccines and drugs against pathogens that pose major public health threats including Covid-19, pandemic influenza, Ebola, HIV, malaria, and TB. The team employs advanced technologies including artificial intelligence to accelerate vaccine development. The team was successful in obtaining regulatory approved for its Covid-19 protein-based vaccine in early October 2021, making it the first recombinant full spike protein in the world to reach this critical milestone. The team had to overcome numerous obstacles, including major supply chain disruptions and local lockdowns, on top of the usual challenges of optimising protein expression, purification and characterisation. Nevertheless, they managed to complete the entire process from initial vaccine design to regulatory approval in just 18 months, making the record for the fastest development through to approval of a recombinant protein vaccine. Whilst not the first Covid-19 vaccines to be approved, Covax19/Spikogen has many promising features including high yield and low cost, temperature stability, strong T and B cell memory, broad cross-protection and excellent tolerability and safety, that may ultimately give it the edge in the highly competitive Covid-19 vaccine arena. Prof. Nikolai Petrovsky is the Research Director of Vaxine Pty Ltd, a Professor of Medicine at Flinders University and an active clinician. He has held multiple large research grants from the US National Institutes of Health and has authored over 200 peer-reviewed research papers. He has won several prestigious awards including an Ernst & Young Entrepreneur of the Year Award in 2010. His recent focus has development of a protein-based vaccine against COVID-19 called Covax-19/Spikogen, which is now approved as both a primary course and 3rd dose booster and forms part of the Iranian national immunization program.
Prof. Stephen Kent is a Professor of Microbiology and Immunology at the Doherty Institute of the University of Melbourne. He trained as an infectious diseases physician, immunologist and vaccine scientist in Melbourne and the USA. He manages a large group of immunologists dedicated to understanding and improving immunity to pandemic viral pathogens such as SARS-CoV-2. Dr Kent remains active in infectious diseases clinical medicine, caring for people infectious diseases and assisting in the clinical development of new vaccine and immunotherapy strategies.
Individuals with potential exposure to SARS-CoV-2 do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication–transcription complex (RTC), in intensively monitored healthcare workers (HCWs) who tested repeatedly negative by PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2, suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae. Dr. Leo Swadling is a research fellow within the division of infection and immunity at UCL. His research focuses on identifying T cell correlates of protection by applying emerging technologies and bioinformatics to the characterisation of T cells at the extremes of controlled or uncontrolled viral infection and post-vaccination. He is funded by the Medical Research Foundation to probe the link between specificity and function of liver-resident memory T-cells in resolved HBV infection and a Rosetree trust/Pears foundation Advancement fellowship to continue his work investigating T cell correlates of protection in abortive versus seropositive SARS-CoV-2 infection.
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) has caused a global pandemic. Two doses of an inactivated SARS-CoV-2 vaccine (CoronaVac) were insufficient to protect against variants of concern (VOCs), while viral vector vaccines remain protective against the infection. Here we conducted a preliminary study to evaluate safety and immunity in adult population who received conventional 2 dosage-regimen of inactivated SARS-CoV-2 vaccine with additional intradermal ChAdOx1 nCoV-19 reciprocal dosage (1:5). Intramuscular ChAdOx1 nCoV-19 boost was also included as a control. Immediate and delay local reactions were frequently observed in the fractional intradermal boost but systemic side effects were significantly decreased compared to the conventional intramuscular boost. The anti-RBD-IgG levels, the neutralizing function against delta variants and T cell responses were significantly increased after boosting with both routes. Taken together, reciprocal dosage of intradermal ChAdOx1 nCoV-19 boost induces similar humoral and cellular immune responses and offers less systemic adverse reactions compared to full dose of intramuscular boosting. These findings provide an effective vaccine management during the shortage of vaccine supply. Dr. Nawamin Pinpathomrat is a lecturer at the Department of Biomedical Science, Medicine at Prince Songkla University. He did his DPhil at the Jenner institute, University of Oxford on pre-clinical development of a viral vector TB vaccine. Interesting fact - in the last year of his DPhil, he unexpectedly ended up being a MasterChef UK Finalist 2018 so do check out his crab dance that has became viral across social media! His current research focuses on safety and immunogenicity of COVID-19 vaccines comparing different regimens and routes of vaccination. Antibody responses are analysed using ELISA and Plaque reduction neutralization tests. ELISpot and flow cytometry are used to observe T cell phenotypes and their effector function.
SARS-CoV-2 infection and vaccination elicit strong CD4+ T cell responses to the viral spike protein, including circulating T follicular helper (cTFH) cells that correlate with the development of neutralizing antibodies. Here we use a novel HLA-DRB1*15:01/S751 tetramer to precisely track spike-specific CD4+ T cells following recovery from mild/moderate COVID-19, after 3 doses of spike-encoding vaccines or after vaccination of previously infected individuals. Infection and primary vaccination induced robust S751-specific CXCR5- and cTFH memory responses in all individuals studied, which were distinct from a subdominant HLA-DRB1*15:01/S236-specific response in the same cohort. Across both infection and vaccination, the S751-specific response is characterised by a restricted TCR repertoire that includes a highly public clonotype. Notably, we observed recall of infection-induced TCR clones by subsequent vaccination and a high degree of clonotypic overlap between CXCR5- and cTFH populations. Overall, this study demonstrates the generation of a stable pool of cTFH and memory CD4+ T cells following infection and vaccination that are efficiently recalled upon spike antigen re-exposure, which may play an important role in long-term protection against SARS-CoV-2 infection. Dr. Jennifer Juno is a T cell immunologist with a particular interest in T follicular helper cells and unconventional T cells. Originally from Canada, she was awarded her PhD in 2014 from the University of Manitoba, where she studied unconventional T cell dysfunction during HIV infection. Following a 2-year post-doctoral fellowship at the Public Health Agency of Canada, she moved to Melbourne to join the Peter Doherty Institute for Infection and Immunity. Jennifer now leads a team focused on harnessing specific T cell subsets to improve immunity against human infectious diseases.
Both infection and vaccination, alone or in combination, induce effective immune responses against SARS-CoV-2. The maintenance of such responses - and hence protection - however may differ. In a large prospective study of UK Healthcare Workers (PITCH, within the larger SIREN study) we previously observed that prior infection impacted strongly on subsequent cellular and humoral immunity induced after long and short dosing intervals of Pfizer-mRNA vaccination. We are now undertaking a longer term study of this cohort over 6-9 months following vaccination and also following a subsequent booster vaccination. We have observed a waning of protection over time post second dose seen most markedly in those without prior infection. The underlying immunologic features of this will be discussed. Prof. Paul Klenerman is trained in medicine at Cambridge and Oxford and specialised in infectious diseases. After a PhD on HIV with Andrew McMichael and Rodney Philips in Oxford, he worked on LCMV in Zurich with Rolf Zinkernagel and Hans Hengartner. He had Wellcome funding to work on T cells and virus infection since then and have looked at T cell responses to CMV and to HCV, including development of vaccines. This includes studies of intrahepatic T cells, which led on to analysing the abundant unconventional T cell populations. In the Covid-19 response he helped integrate immunology studies from across Oxford, and established PITCH, a large collaboration focusing on T cell responses in UK Health Care Worker. He holds a chair at Oxford, working at the Translational Gastroenterology Unit and at the Medawar Building (Pathogen Research).
Prof. Antonio Bertoletti is an expert in the field of viral hepatitis, with a specific interest in the immunopathogenesis of HBV infection and is a Professor at the Emerging Viral Disease Program at Duke-NUS Medical School, Singapore. His current research is focus on the development of new immunological based therapies (TCR-redirected T cells) for the treatment of HBV chronic infection and Hepatocellular carcinoma. In 2020, after the start of the COVID-19 pandemic, his laboratory has been actively involved in the characterization of SARS-COV2 specific T cell immune response in infected and vaccinated individuals.
Yanchun Peng is a senior Postdoctoral researcher working at the Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford. After completing her undergraduate studies and then teaching in China, she moved the UK, first working as a research assistant, then completing her DPhil and remaining as a Postdoc in the research group led by Prof. Tao Dong since 2005. She has acquired extensive experience and skills in human T cell immunology with the major focus of her research being on evaluating anti-viral efficacy of virus-specific T cells and the interaction of host and virus in HIV infection, HIV/HCV co-infection and influenza infection. Her recent interest is to study the Viral OncoProtein(VOP) and tumor Specific Protein(TSP) specific T cell responses in virus associated cancer (ie HBV/HCC; EBV/NPC and HPV/CC) by combining cutting edge technologies (such as multi-colour Flow cytometry, Mass Cytometry (CyTOF), single cell RNASeq with SmartSeq 2 and 10X Chromium) with my expertise in isolating, expanding and characterising virus/tumor-specific T cells in vitro. Being equipped with these well-established platforms, skills and knowledge in human T cell immunology, she swiftly switched my work from cancer to COVID-19, as the pandemic started in 2020: exploring the role of SARS-CoV-2 -specific T cells in COVID-19 infection.
Questions addressed by the expert panelists include: 1. As viral immunologists you have all rapidly moved from your own area of expertise to characterising the immune response to SARS-CoV-2. What has most surprised and intrigued you in the study of the virus? 2. If SARS-CoV-2 variants totally evade vaccine-induced neutralising antibodies, can vaccine-induced memory T cells still contribute to host protection? 3. What insights do you have regarding the immune response in the context of re-infection of the same or different variants? Our sincere thanks to the expert panel for their contribution: Prof. Eui-Cheol Shin (KAIST, Korea) Dr. Nina Le Bert (Duke-NUS Medical School, Singapore) Prof. Tao Dong (University of Oxford, UK) Dr. Yanchun Peng (University of Oxford, UK)
In this talk, Dr Broderick introduces us to the INO-4800 SARS-CoV-2 DNA vaccine and shares Phase I and II clinical trial data. The vaccine is shown to induce broad antibody and persistent CD8 T cell responses (including T cell responses targeting Variants of Concern). Kate Broderick received her Ph.D. from the University of Glasgow, Scotland, and conducted post-doctoral research at the University of California, San Diego. She joined INOVIO in 2006, where she currently leads a diverse research group focused on enhanced delivery techniques for gene-based therapeutics as the Senior Vice President, Preclinical R&D. She is a co-inventor on multiple patents related to DNA vaccine delivery and has served as a principal investigator on grants, awards, and contracts from leading government agencies and not-for-profit organizations, including the Bill and Melinda Gates Foundation, the National Institutes of Health, the U.S. Department of Defense, the Small Business Innovation Research program, and including a $56M award from the Coalition for Epidemic Preparedness Innovations (CEPI). In 2018, Dr. Broderick was named Business Women of the Year by San Diego Business Journal. Her particular area of focus is infectious diseases with an emphasis on emerging targets and those from a bio-threat potential. Most recently, Dr. Broderick led the teams that brought the first in human Lassa fever vaccine into the clinic as well as advanced the development of a DNA vaccine for the MERS virus and she is responsible for driving the development of a DNA vaccine for COVID-19.
In this presentation, Dr. Sette provides evidence that there are robust CD4+ and CD8+ T cell responses detected in uncomplicated SARS-CoV-2 convalescent cases; that reactivity is reproducibly detected in non-exposed subjects; specific CD4 and CD8 T cells targets in COVID-19 patients were identified; in acute and severe infection it appears that the speed of the adaptive response is key to protective immunity; that T cell responses are durable over at least 8 months and that there is negligible impact of SARS-CoV-2 variants on T cell responses. Alessandro Sette has devoted more than 35 years in biotech and academia to understanding and measuring immune responses, and developing disease intervention strategies against cancer, autoimmunity, allergy, and infectious diseases. Dr. Sette’s laboratory is the world leader in the study of the specific structures, called epitopes, that the immune system recognizes. Dr. Sette has overseen the design and curation efforts of the national Immune Epitope Database (IEDB), a freely available, widely used bioinformatics resource. The IEDB catalogs all epitopes for humans and experimental animals for allergens, infectious diseases, autoantigens and transplants, and includes epitope prediction tools to accelerate immunology research around the world. Dr. Sette’s lab uses knowledge of epitopes to define the hallmarks of a beneficial immune response associated with effective vaccines, as opposed to immune responses that are ineffective or that cause harm. The laboratory’s infectious disease interests include SARS CoV2, dengue, Zika Chikungunya, herpesviruses, poxviruses, lassa fever, HIV and hepatitis viruses, and bacterial pathogens such as tuberculosis and bordetella pertussis. Our investigations outside infectious disease include allergic asthma and Parkinson’s disease. Dr. Sette is a Doctor in Biological Sciences from the University of Rome and did postdoctoral work at the National Jewish Center for Immunology and Respiratory Medicine in Denver, Colorado. In 1988, Dr. Sette joined the newly founded company Cytel, in La Jolla, and was also appointed adjunct assistant professor at The Scripps Research Institute. He founded Epimmune in 1997, where he served both as Vice President of Research and Chief Scientific Officer until 2002, when he joined LJI as Head of the Division of Vaccine Discovery. He also heads the Center for Infectious Disease at LJI.
In this short talk, Edmund presents an introduction to ProImmune, the tools available for monitoring antigen-specfic T cell responses focusing on Pro5 MHC Pentamers and MHC class II tetramers, and through presentation of case studies, the application of these sensitive technologies to characterise the frequency of SARS-CoV-2 and other viral-specific T cell responses. Edmund Neo is ProImmune’s Immunology Sales Specialist located in Singapore representing the company in the Asia-Pacific Region. Edmund graduated from the National University of Singapore and worked with Abbott Laboratories before joining ProImmune in 2019. He has played a pivotal role in the team supporting customers across his region.
In this talk we learn that SARS-CoV-2-specific PD-1+CD8+ T cells are not exhausted, but functional; that they are sustained for at least 8 months, as well as learning about the development of SARS-CoV-2-specific stem cell-like memory T cells (TSCM). Eui-Cheol Shin received his M.D. (1996) and Ph.D. (2001) from Yonsei University, Seoul, Korea, and his postdoctoral training from National Institutes of Health, Bethesda, Maryland, USA. Then he joined Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea in 2007, where he is currently a professor. His laboratory, the Laboratory of Immunology and Infectious Diseases, focuses on the research of T cell responses in human viral disease and cancer.
In this talk we learn how quantity and early induction of virus-sepcfici T cells is directly associated with mild disease; how higher functionality of SARS-CoV-2 specific T cells is found in assymptomatic cases of SARS-CoV-2 infection and how how early detection of SARS-CoV-2 specific T cells correlates with the the onset of vaccine efficacy. Nina Le Bert is currently a Senior Research Fellow in the Emerging Infectious Diseases Programme of DUKE-NUS Medical School in Singapore. She earned her Master of Science degree at the Free University of Berlin in Germany, and her PhD from the University College London UK in 2011, working in the field of human innate and adaptive immunity. In 2014, she joined Professor Antonio Bertoletti’s lab, where she is investigating the virus-specific B and T cell responses during Hepatitis B Virus (HBV) infection to better understand their role in protection and/or liver pathology. Since the outbreak of the COVID19 pandemic, she is involved in studies of SARS-CoV-2 specific T cells.
Katie Ewer is Associate Professor and Senior Immunologist at the Jenner Institute, University of Oxford where she leads a program of exploratory immunology studying vaccine-induced immunity to malaria and outbreak pathogens, such as Ebola, MERS and now SARS-CoV-2. A key aim of her research is focused on characterizing T cell responses induced by vaccination with viral vectors. During the COVID-19 pandemic, she played a pivotal role in the clinical trials in an effort to find a vaccine, which has led to the successful roll-out of the first approved SARS-CoV-2 vaccine, ChAdOx1 in partnership with AstraZeneca in December 2020. In this talk she explains how the vaccine was developed in record-breaking time, the characterisation of the immune responses to the vaccine and the forthcoming challenges still faced including the question of booster doses, manufacturing and supply and how we respond to the next inevitable pandemic.
For forty years, I’ve been researching therapeutic antibodies with a particular interest in effector functions of the Fc region. With Herman Waldmann and Greg Winter, I took several of the early engineered antibodies into the clinic. We realised that activation of immune effector systems is not always desirable, but found that mutations designed to reduce binding to Fc receptors (e.g. ‘LALA’, aglycosyl) were not completely effective. At mAbsolve we have screened hundreds of new variants and identified some which are completely devoid of binding to Fc receptors. Receptor binding is one thing, but cellular activity is more important. The modest binding levels of previous variants translated to quite substantial cellular activity, but our new variants remained completely negative. The risk of unwanted immunogenicity is always a concern when proteins are modified, but the variants gave no increased risk signals either in silico or in vitro. The optimised Fc variant is comparable with wild type IgG for binding to FcRn, stability and manufacturability and is available for licensing.
Epitope discovery is a crucial element in the development of vaccine candidates and drug therapeutics. In the Immune-oncology space, identifying neoepitopes and tumor- associated antigens provide new targets for cancer diagnostics and enable the tracking of patient responses to treatment. Whereas, monitoring T cell responses to gene therapy vectors and transgenes is important in evaluating efficacy and safety of the treatment. ProImmune provides industry-leading tools for antigen characterization, epitope mapping and immune monitoring. Novel epitopes can be identified by the highly sensitive ProPresent® Antigen Presentation assay, which utilizes LC/MS/MS to determine peptides that are naturally processed and presented. Additionally, the REVEAL® HLA-peptide binding assay eliminates the need for precious patient samples and allows for the screening of multiple proteins for potential T cell epitopes in a matter of weeks. Once the epitopes have been identified, they act as important markers for tracking treatment effectiveness in clinical immune monitoring programs. IFN-γ ELISpots serve as a robust measurement of antigen specific immune responses following patient vaccination and/or treatment, while antigen-specific CD4+ T and CD8+ T cell responses can be enumerated by the ProT2® MHC-Tetramers and Pro5® MHC-Pentamers, respectively. In this presentation, case studies will be shared that detail how ProImmune’s integrated platform has identified novel epitopes in the immune-oncology and gene therapy field and enabled the detection and measurement of functional T cell responses against those epitopes in the clinic.
Immunotherapy based on adoptive transfer of T cells engineered to express HLA class I restricted T-cell receptor (TCR) or a chimeric antigen receptor (CAR) targeting tumor antigens has generated impressive results in treating some cancers. T cells are, however, also essential for the control of viral infections, particularly in persistent viral infections like HBV that is characterized by defect of antigen specific T cells. We have built a library of virus-specific TCRs (mainly HBV-specific) that we use both in vitro and in vivo to engineer lymphocytes able to target both HBV infected hepatocytes and HBV-related liver tumor. However, while anti-cancer therapy seeks an efficient killing of cancer cells, antiviral therapy should maximize the suppression of viral replication with limited organ damage, particularly in viral infection targeting organs that are indispensable for life (as the liver). I will discuss the strategies, such as time controlled TCR expression or modification of T cell function through the use on antisense nucleotides targeting T effector proteins, that are utilized in our laboratory to engineer antiviral lymphocytes with limited lysis ability for chronic viral infection treatment.
Hilario Ramos, Molecular Templates, discusses the use of Egineer Toxin Bodies (EBTs) - molecules with an antibody domain for targeting that is genetically fused to a de-immunized Shiga-like toxin A subunit which drives apoptotic cell death. These molecules can drive cell death by apoptosis, and Hilario discusses novel uses for EBTs of delivering antigenic peptides to tumours, in a novel way to make the tumour cells visible to the immune system. Hilario highlights the use of ProImmune's Pro5® MHC Class I Pentamers in tracking Antigen Specific CMV CD8 T cells in order to help build their CTL models.
A great introduction to the chimanzee adenoviral vector platform that ultimately went on to be developed into the frrst approved vaccine against SARS-CoV-2.
Immunogenicity is one of the most complex issues to address in drug design and development and requires intelligent application of integrated platforms to mitigate the risk to your biologic. In this talk I will present case studies to illustrate the range of solutions that ProImmune provides including DC-T/T cell proliferation assays for lead selection/optimization, MAPPS assays for characterization of antigen presentation; HLA-peptide binding assays to characterize individual epitopes & undiluted whole blood cytokine storm assays. Jeremy gained his DPhil. under the supervision of Prof. Kathryn Wood from the University of Oxford developing gene therapy strategies to induce immunological tolerance in transplant recipients. Jeremy joined ProImmune in 2001 to generate a new class of MHC multimer staining reagents. For the past 19 years as ProImmune's Director of Sales, he has led the sales team in a growing business, focusing on developing and implementing innovative technologies that radically improve our understanding of immune responses.
Oesophageal cancer (OC) causes over 0.5 million deaths worldwide each year. Current therapeutic regimens focus on chemo-radiotherapy prior to surgery, however, only 20-30% of patients respond to treatment. Therefore, new treatments are badly needed. Natural killer T (NKT) cells are innate T cells that recognize lipid antigens presented by CD1d molecules. We enumerated circulating NKT cells in OC patients and control subjects by flow cytometry using monoclonal antibodies and CD1d tetramers loaded with glycolipid antigens. This allowed for the detection of invariant NKT cells, which possess invariant T cell receptor (TCR) α-chains reactive with α-galactosylceramide and mediate anti-tumour immunity in mice and humans, as well as novel NKT cells that recognise other glycolipids. We found that invariant NKT cells were depleted and functionally-impaired in patients with OC. In contrast, NKT cells reactive against sulfatide and tetramyristoyl-cardiolipin (TCL) were present in significantly higher numbers in OC patients compared to controls. Most of these cells expressed γδ TCRs, specifically the Vδ1 subset. TCL induced the production of transforming growth factor-β by expanded Vδ1 T cells in vitro in a CD1d dependant manner. Supernatants of Vδ1 T cells activated with sulfatide or TCL inhibited degranulation by invariant NKT cells. These data suggest that invariant NKT cells protect against OC but other CD1d-restricted NKT cells may promote tumorigenesis in these patients. These findings have important implications for cellular therapies involving invariant NKT cells, which are currently being tested in clinical trials for multiple cancer types.
Sequence variations in CMV UL40-derived peptides control NK activation through HLA-E presentation.
Edmund Neo is ProImmune’s Immunology Sales Specialist located in Singapore representing the company in the Asia-Pacific Region. Edmund graduated from the National University of Singapore and worked with Abbott Laboratories before joining ProImmune in 2019. He has played a pivotal role in the team supporting customers across his region.
Upon kidney transplantation, de novo donor-specific antibodies (dnDSA) can be triggered by immunogenic polymorphic amino acids (AA) configurations on mismatched donor HLA. These configurations, called eplets, are theoretically defined and require experimental verification to establish whether allo-antibodies can actually bind to these specific polymorphic residues. Human HLA-specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we aimed to generate recombinant human HLA-DR specific mAbs for the verification of HLA-DR eplets. We isolated single HLA-DR-specific memory B cells from peripheral blood of immunised individuals (n = 3) using HLA class II tetramers. Upon cell sorting, an average of 5 HLA-antibody positive B cell clones (range 2-11) were isolated per individual. The clones obtained from a single individual differed in clonality and specificity. From the HLA-specific memory B cells we generated recombinant human HLA-DR-specific mAbs (n = 5). The mAbs were screened with Luminex single antigen bead assay. The AA sequences of the reactive HLA antigens were compared using the software program HLA-EMMA to identify uniquely shared AA within 3-3.5Å, which are referred to as functional epitopes. This reactivity analysis led to identification of three configurations i.e. 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognised respectively by HLA-DRB1*01:01, HLA-DRB1*04:01, and HLA-DRB1*07:01 antigen-reactive mAbs. The former two correspond to eplets 70QA and 31FYY and can now be considered as antibody-verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody-verified. The verification of these eplets will contribute to the implementation of eplet matching in allocation systems aiming to prevent the development of dnDSA. The generated recombinant human HLA-DR-specific mAbs can also be used in functional studies to provide insight in the respective roles of HLA-specific IgG antibodies in causing graft damage.
Since the early days of the development of the CRISPR Cas9 gene editing technology there have been sporadic speculations that immune responses to Cas9 (which is of microbial origin) could have consequences for clinical applications. Since late last year, three studies have presented experimental evidence for T- and B-cell responses to Cas9. These results have prompted considerable commentary, in both the scientific and popular press, marking Cas9-immunogenicity as a major obstacle to bringing CRISPR genome-editing to the clinic. The studies on Cas9 immunogenicity have brought attention to a critical element in the eventual clinical application of Cas9-mediated gene editing. In this presentation I will provide results of recent studies using a broad array of assays including ELISAs to detect anti-Cas9 antibodies, T-cell proliferation assays to identify immune-hot spots on Cas9 and MHC Associated Peptide Proteomics. The experimental data demonstrate the need for fit-for-purpose assays, statistical methods and reagents that will be necessary for obtaining data that is sufficiently reliable to make decisions for drug development and licensure. The broad outline of my presentation is as follow: • Unique challenges while assessing the immunogenicity risk to Cas9 • The need of validated reproducible assays that are fit-for-purpose • The need for well characterized reagents and for controlling contaminants and impurities • Model systems for evaluating the immunogenicity of Cas9
Dr Amy Flaxman is part of the Oxford Vaccine Group, Jenner Institute, University of Oxford. For her post-doctoral research she has worked on both pre-clinical studies developing vaccines for outbreak pathogens, such as Ebola, and clinical trials for outbreak pathogens, including MERS and Ebola. She is currently investigating immune responses to the Oxford ChAdOx1-nCoV vaccine for SARS-CoV-2 that was in Phase III clinical trials but is now approved. She leads the lab team carrying out antibody testing post-vaccination. Here, she is interested in the differences in antibody responses induced over time with different doses of vaccine, in different age groups and how this changes with administration of a booster vaccine.
The rationale for therapeutic targeting of Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αβ T cells. Nonetheless, insights regarding Vδ2+ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to challenging scarcity of these cells in surgical specimens. αβ T cell phenotypic alterations occurring in the tumor bed are detectable in periphery and correlate with adverse clinical outcome. Thus, we sought to determine whether Vδ2+ T cells phenotypic changes can be detected within breast cancer patients’ peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients’ peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ T cells displaying phenotypes of exhausted senescent T cell associated with lymph node involvement. Thereby, our results support Vδ2+ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ T cell anti-tumor activity.
The a priori T cell repertoire and immune response against SARS-CoV-2 viral antigens may explain the varying clinical course and prognosis of patients having a mild COVID-19 infection as opposed to those developing more fulminant multisystem organ failure and associated mortality. Using a novel SARSCov-2-specific artificial antigen presenting cell (aAPC), coupled with a rapid expansion protocol (REP) as practiced in tumor infiltrating lymphocytes (TIL) therapy, we generate an immune catalytic quantity of Virus Induced Lymphocytes (VIL). Using T cell receptor (TCR)-specific aAPCs carrying co-stimulatory molecules and major histocompatibility complex (MHC) class-I immunodominant SARS-CoV-2 peptide-pentamer complexes, we expand virus-specific VIL derived from peripheral blood mononuclear cells (PBMC) of convalescent COVID-19 patients up to 1,000-fold. This is achieved in a clinically relevant 7-day vein-to-vein time-course as a potential adoptive cell therapy (ACT) for COVID-19. We also evaluate this approach for other viral pathogens using Cytomegalovirus (CMV)-specific VIL from donors as a control. Rapidly expanded VIL are enriched in virus antigen-specificity and show an activated, polyfunctional cytokine profile and T effector memory phenotype which may contribute to a robust immune response. Virus-specific T cells can also be delivered allogeneically via MHC-typing and patient human leukocyte antigen (HLA)-matching to provide pragmatic treatment in a large-scale therapeutic setting. These data suggest that VIL may represent a novel therapeutic option that warrants further clinical investigation in the armamentarium against COVID-19 and other possible future pandemics.
Dr Lea Bartsch, of Massachussets General Hospital and Harvard Medical School, discusses her approach of using Pro5 Pentamers and ProT2 Tetramers in HBV infections. Explaining the T cell dysfunction caused by HBV that leads to the viral prevalence, with little ex vivo data avaliable on the function of HBV specific T cells, the use of Pentamers and Tetramers have enabled their group to chracterize the functionality of these T cells.
Dr. Natasa Strbo from the University of Miami, discusses the use of ProImmune's Pro5 Pentamer technology in her work involving SARS-CoV-2 protein based vaccine development. Their vaccine is a cell based Heat shcok protein (Gp96-Ig) vaccine, which serves as an adjuvent as well as the antigen carrier (SARS-CoV-2 Spike protein). They used two known immunogenic epitopes from the S protein and sucessfully stained the vaccinated mouse cells with ProImmune Pro5 Pentamers, and analysed the Pentamer positive cells for intracellular cytokine secretion. With the use of Pentamers they showed an induction of S protein specicfic CD8 Tcells in lung and spleen murine cells.
Epitope discovery and immune monitoring is a crucial element in the development of vaccine candidates and drug therapeutics. ProImmune provides industry-leading tools for antigen characterization, epitope mapping and immune monitoring. Novel epitopes can be identified by the highly sensitive ProPresent® Antigen Presentation assay, which utilizes LCMS/MS to determine peptides that are naturally processed and presented. Additionally, the REVEAL® HLA-peptide binding assay eliminates the need for precious patient samples and allows for the screening of multiple proteins for potential T cell epitopes in a matter of weeks. Once the epitopes have been identified, they act as important markers for tracking treatment effectiveness in clinical immune monitoring programs. IFN-γ ELISpots serve as a robust measurement of antigen specific immune responses following patient vaccination and/or treatment, while antigen-specific CD4+ T and CD8+ T cell responses can be enumerated by the ProT2® MHC-Tetramers and Pro5®MHC-Pentamers, respectively. I will be sharing case studies about the success of the HCV Vaccine Trials at Oxford University that has deployed our Pro5®MHC-Pentamers and how ProImmune’s integrated platform that has identified novel epitopes in the immune-oncology and gene therapy field that enabled the detection and measurement of functional T cell responses against those epitopes in the clinic.
In viral hepatitis, activation of bystander CD8+ T cells has been reported, but their roles have not been elucidated. Recently, we reported that bystander CD8+ T cells contribute to the liver injury in acute hepatitis A virus (HAV) infection (Immunity 2018, 48:161). In patients with acute HAV infection, CD8+ T cells specific to unrelated viruses, including CMV, EBV, and influenza virus, become activated by IL-15. Activated bystander CD8+ T cells exert innate-like cytotoxicity triggered by activating receptors NKG2D and NKp30 without TCR engagement. Importantly, the severity of liver injury is associated with activation and innate-like cytotoxicity of HAV-unrelated virus-specific CD8+ T cells, but not the activation of HAV-specific T cells, indicating that bystander-activated CD8+ T cells are implicated in host injury during acute viral hepatitis. More recently, we also examined tissue-resident memory (Trm) phenotypes of hepatitis virus-specific and non-hepatotropic virus-specific CD8+ T cells among liver sinusoidal mononuclear cells (J Hepatol 2020, 72:1170). There are two different Trm-like CD8+ T cell populations, CD69+CD103+ and CD69+CD103-. Interestingly, CD69+CD103+CD8+ T cells include only hepatitis virus-specific cells whereas CD69+CD103-CD8+ T cells include both hepatitis virus-specific and non-hepatotropic virus-specific cells, indicating that tissue-tropism of viruses is associated with tissue-resident features of CD8+ T cells in the liver.
Dr. Aaron Mansfield of Mayo Clinic Cancer Center discusses the current immunotherapy treatments for patients with mesothelioma, the relatively low tumour mutation burden in those patients and alternatives to non-synonymous point mutations as sources of neo-anitgens. The use of ProImmune REVEAL technology allowed him to compare the MHC binding scores of selected peptides from chromosomal rearrangements, of usually non-coding DNA to discover if any of these peptides were neo-antigenic.
Dr. Emilee Knowlton of ProImmune describes the tools for epitope discovery and immune monitoring, providing case studies and considerations for both pre-clinical and clinical
Dr. Yeojun Yun discusses Synteka Bio's NEOscan™, a neoantigen prediction algorithm used to improve the selection and prioritization of neoantigens for inclusion in cancer therapies.
Integrated platforms can be used to mitigate immunogenicity risk and characterize immune responses during the drug design and development stages. ProImmune offers mutational activity mapping for optimal protein design, DC-T/T cell proliferation assays for biologic lead selection/optimization, a Mass Spectrometry assay for characterization of antigen presentation; HLA-peptide binding assays to characterize individual epitopes & undiluted whole blood cytokine storm assays.
Dr Lea Bartsch from Massachusetts General Hospital discusses her work with HBV, in trying to better the understanding of the immunological alterations in order to improve treatment strategies for HBV infection. She highlights the use of ProImmune's custom ProT2 Class II Tetramers in tracking HBV antigen specific CD4 T cells to validate new HBV epitopes and analyze the different CD4 T cell responses in chronic vs. acute infections.
Dr. Amy Rosenberg of CDER, FDA discusses the clinical context for the reappraisal of immunogenicity of therapeutic proteins that are being used to treat COVID-19. She highlights the importance of understanding the autoinflammatory or autoimmune response following infection SARS-CoV-2 and its potential to increase immunogenicity to self proteins leading to autoimmune and autoinflammatory disorders. This indicates the high probability that this could lead to higher immunogenicity in patients to therapeutics sued to treat SARS-CoV-2.
Dr Tomer Hertz and Dr Amir Aharoni of Ben Gurion University of the Negev, discusses their new approach for therapeutic protein de-immunization, reducing ADA's, whilst still preserving drug efficacy. Their strategy focuses on combining computational and experimental immunology with protein engineering. Their case study on Adalimumab (Humira) and Ipilimumab shows how their method of first identifying the potential immunogenic CD4 hotspots on the drug by computational methods, and then secondly using Yeast Surface Display protein engineering to eliminate these hotspots but maintain the WT activity by using a high throughput screening approach, results in a still active but less immunogenic variant.
Dr. Nina Le Bert from Duke NUS, discusses her recent involvement in the study of SARS-CoV-2 specific T cell immunity, specifically looking at the induction and persistence of T cells using an overlapping peptide library of specific proteins in SARS-CoV-2 to test convalescent patients' IFN-gamma responses to these peptides. Using a cohort of individuals who were infected and recovered in 2003 with SARS, and a similar approach of overlapping peptides from SARS-CoV, she is able to show there are still persistent ex vivo T cell responses. Importantly, she also highlights the cross reactivity of NP peptides from SARS-CoV and SARS-CoV-2.
Dr. Kellie Smith from Johns Hopkins, discusses her group's global study of the associations of neoantigen specific T cell responses with clinical outcomes to checkpoint blockades in cancer. The talk outlines the use of their MANAFEST T cell receptor sequencing technology, and by sequencing the CDR3 region, they are able to track clones that recognise the same antigen of interest. Showing that with lower clonality there is an association with higher residual tumour burden in patients. Pro5 Pentamers are also shown being used as an accurate way to validate their findings, by using them to track the neoantigen specific CD8 T cells. She also highlights the use of single cell RNA-seq in determining the phenotype of the clones in responding and non-responding patients.
Professor Tao Dong from the University of Oxford discusses her latest work in memory T cell responses in convalescent SARS-CoV-2 patients in the UK, highlighting the information from her publication in Nature Immunology and the use of ProImmune Pro5® Pentamers in her work.
Dr. Tim Hickling discusses the importance in the application of immunogenicity prediction and mitigation in the production of biotherapeutics. He outlines some of the current methods for predicting immunogenicity and highlights the need for better accuracy in predictions.
Professor David Withers from the University of Birmingham discusses his recent work in tracking dynamic changes of lymphocytes within tumours, to understand and characterise the phenotype of cells entering, leaving and being retained in the tumour through the use of the transgenic Kaede Photoconvertible mouse model. He also discusses the use of Single Cell RNA-seq to capture the heterogeneity within the tumour infiltrating lymphocyte population, and describing the use of ProImmune Pro5® Pentamers to track tumour neo-antigen specific CD8 T cells.
Antigen processing and presentation by HLA class II is a complex process and current in silico predictions are not able to account for all the factors involved in immunodominance/immunogenicity as they focus only the binding component of the process. Generation of large training datasets from single HLA class II allele cell-based or cell-free MAPPS-like platforms is enabling development of algorithms for identification of naturally processed ligands. The combination of binding and processing is a more reliable approach to the identification of viable CD4+ T cell epitopes, and to rank molecules. In silico prediction still needs to be part of a more complex immunognicity risk asessment strategy.
Katerina describes the process of codon optimization and the role that it plays in a protein's rate of translation, expression and conformational properties. She describes the presence of synonymous mutations in various diseases and the immunogenicity implications relating to those mutations. She uses Factor IX as a model to describe the workflow to generate variants using CoCoPUTs and the evaluation of those variants for protein expression, confirmational differences, peptide presentation and many more attributes.
Patrick describes the Children’s National cellular therapy program for treating viral infections following transplant surgeries. He discussed the generation of donor-derived virus specific T cells from cord using either an adenoviral vector or peptide mixes and the recognition of atypical epitopes by these cells. The team has treated over 80 patients with virus specific T cells with between 70-80% response rate.
Paul discusses the challenges facing the identification and diagnosis of Lyme disease. He describes the life cycle stages of the carrier, Borrelia burgdorferi sensu lato species, as well as the incidence and transmission of Lyme disease in the United States. There are differential expressions of antigenic surface proteins that occur due to bacterial transcriptional changes and throughout the course of the disease. Highly reactive serum was screened for responses against 21 different proteins using 80 overlapping peptides in the ProArray Ultra linear B cell epitope mapping tool to improve the specificity and accuracy of current diagnostic methods.
Rao describes Multiple Myeloma, a plasma cell cancer and the biomarkers that can be used to classify and monitor the immune response through the progression of the disease. They Harvard group looked at myeloid derived suppressor cells, plasmacytoid DC, T-regulatory cells, TH17 cells and B cell subpopulations in the interrogation of the cancer. There was a humoral immunity deficiency, increased PD1 expression on effector cells and increased levels of PDL1 expression detected in the multiple myeloma patients.
Vibha discusses a proactive risk based approach to assessing immunogenicity in early development. She describes a suite of in silico risk assessment tools in combination with in vitro methods to evaluate immunogenicity or product attributes and how, when used in combination, those methods can reduce false positive identification. The stages of when to implement and report the data from the risk assessment tools is described as well as the current challenges the FDA reviewers face when evaluating the data package.
Beatriz discusses the use of cancer vaccines to transition ‘cold’ tumors in patients to ‘hot’ tumors. The UPenn group used Next Gen Sequencing to catalog tumor mutations and generated autologous dendritic cell vaccines to evaluate the immunogenicity of missense mutations and define the nature of patient T cells specific for melanoma neoantigens. They found that that immunological ignorance of clonal neoantigens is the basis for ineffective T cell immunity to melanoma and that therapeutic vaccination as an adjunct to checkpoint inhibitor treatment, should be considered to increase the breadth and diversity of neoantigen-specific CD8+ T cells.
Karen discusses how to measure antigenic diversity of B and T cell immune responses and how the structural components of those antigens drive immunogenicity. She describes technologies such as custom protein-peptide microarrays to detect antibodies; a mass spec based program called MHC TreASUre Hunt for immune peptidome discovery; and Molecular Dynamic Modeling to model peptide MHC interactions based on existing crystal structures to help develop computational systems to predict TCR.
Jonathan Silk is the Head of Cell Research at Adaptimmune (www.adaptimmune.com), a biotechnology company located in Abingdon and Stevenage, UK, and Philadelphia, PA, USA. Adaptimmune is a world leader in the TCR T-cell therapy space, utilizing engineered Specific Enhanced Affinity T-cell receptor-expressing T-cells (SPEAR T-cells) to target both solid and hematologic cancer types. Jonathan and his team are investigating Next Generation SPEAR T-cells, to improve the function of T-cells for adoptive T-cell therapy, including overcoming immune-resistance mechanisms. Jonathan received his BSc in Biochemistry from the University of Kent at Canterbury (UK), with an industrial placement at Merck Sharp and Dohme (Harlow, UK). He obtained a PhD in Immunology studying at the MRC Clinical Sciences Centre (Imperial College London, UK). Subsequently he worked with Professor Vincenzo Cerundolo at the University of Oxford, UK, as a Post-doctoral Scientist, developing a number of cancer immunotherapy research programmes including the use of ligands for invariant NKT-cells as adjuvants and investigating mechanisms of tumour escape.
An overview as to the current strategies in place at GSK to develop novel vaccines aagainst emerging diseases.
Genome editing of T cells has broadened the clinical scope of chimeric antigen receptor (CAR) treatments to include allogeneic T cells from mismatched donors, previously constrained in their use due to HLA barriers and allo-recognition. Initial proof-of-concept applications are underway using transcription activator-like effector nuclease (TALEN) edited ‘universal’ T cells devoid of TCRαβ expression and depleted of CD52 - the target antigen for the lymphodepleting antibody Alemtuzumab. While efficient, editing effects were unlinked, resulting in variable yields of heterogenous T-cell populations, complicating cell dosing strategies. Here, a novel ‘terminal’ vector platform was developed, that couples transgene expression with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 scission effects through precise incorporation of sgRNA element(s) into the ∆U3 3’ lentiviral long terminal repeat (LTR). Transient delivery of Cas9 mRNA by electroporation to ‘terminal’ transduced primary T cells resulted in highly efficient TRAC locus modification yielding homogenous CAR+ (greater than 96%) TCR- (greater than 99%) cells capable of robust in vivo anti-leukaemic effects. The strategy has now been extended to the simultaneous disruption of TCR and HLA class I genomic targets resulting in efficient multiplexed effects. While these approaches rely on nuclease mediated cleavage and cell mediated repair of double standed DNA (dsDNA) breaks, the associated risk of chromosomal translocations is yet to be fully elucidated. Base editing through targeted chemical deamination circumvents dsDNA breaks, and instead offers the possibility of seamless nucleotide conversion for the disruption of coding sequence. sgRNA sequences targeting regions of TCRα and β genes, B2M and CD52 genes were delivered to primary T cells. Transient delivery of codon optimized base editor 3 (coBE3) resulted in high levels of ‘on-target’ base conversion, with protein knockout comparable to that achieved by spCas9 mRNA, while chromosomal translocation events remained undetected using PCR based screening. While additional characterization of CAR T cells with multiple deamination mediated edits is underway, these processes are scalable and amenable to adaption for GMP-compliant manufacturing, and provide a route to translational application.
Human cytomegalovirus (HCMV) frequently reactivates after hematopoietic stem cell transplantation (HSCT). Antiviral medication is not universally effective and has side effects that prohibit prophylaxis and limit pre-emptive use. Adoptive transfer of CMV-specific T lymphocytes can protect patients at risk of CMV disease, but such T cells are difficult to obtain if the donor is CMV-negative. Transferring the T cell receptor (TCR) genes of CMV-specific T cell clones to donor T cells will convey CMV specificity to those cells. But if αβ T cells receive another αβ TCR, both TCRs may form mixed heterodimers. This problem can be solved by expressing αβ TCRs in easily available γδ T cells from the donor. Although HCMV-specific CD4+ T cells are known to play a critical role in controlling HCMV and maintaining immunity, possibilities of their transgenic generation have not been explored. Here we express CMV-specific TCRs from CD4+ T cell clones in T cells from CMV-negative donors. We established a panel of ten CMV-specific MHC class II-restricted TCRs targeting seven distinct epitopes of phosphoprotein 65 (pp65) and immediate-early protein 1 (IE-1) presented by a wide range of MHC II allotypes. These TCR genes were retrovirally transferred into γδ and αβ T cells from CMV-negative donors. The resulting T cells specifically recognized HLA-matched CMV-infected dendritic cells with comparable effectiveness, but backgrounds due to alloreactivity were even lower in γδ T cells than in αβ T cells. Experiments with inactivated virus showed that pp65 was mainly delivered as part of the virions, whereas IE-1 was endogenously expressed and presented to CMV-specific T cells by infected cells. Our results identify γδ T cells that transgenically express MHC II-restricted TCRs as promising immunotherapeutic agents against CMV infection.
The ability to clear many intracellular infections is likely to require a consorted immune response that includes CD8+ T cells. Although a large number of approaches have been taken to generating such responses in humans, achieving the magnitude and functionality of natural viral infections has been difficult. The most promising approach published to date is to employ a heterologous prime-boost, usually using two replicating or non-replicating viral vectors. The Jenner Institute has extensively used a replication-deficient adenovirus followed by a replication-deficient pox virus, and achieve ELISPOT levels of balanced CD4+ and CD8+ T cells in the many thousands of spot forming units per million PBMCs. Vaccitech has in-licensed programs based on the use of this strategy to control chronic HBV and chronic HPV. The progress of these programs will be discussed.
Dr. Butler is Assistant Professor at the Princess Margaret Cancer Centre Tumor Immunotherapy Programme where is he is the Clinical Head of the Immune Monitoring Team. Before joining he was an Instructor in Medicine at Harvard Medical School and Clinical Fellow in Medicine at the Dana Farber Cancer Institute. Dr. Butler's expertise includes clinical trials in a variety of immune therapies including adoptive T cell therapy and checkpoint blockade.
The central role of T cell responses to clinical successes in immuno-oncology has been well appreciated, yet few technologies have been designed to exclusively generate effective and sustained T cell responses in vivo. The DPX platform is a unique oil based formulation that entraps its components and facilitates active uptake by antigen presenting cells at the site of injection. This unique presentation to the immune system results in robust and sustained T cell responses. The lead clinical product, DPX-Survivac, employs the DPX platform to induce T cells targeting the shared tumor associated antigen survivin, which is overexpressed in many human malignancies. DPX-Survivac has been demonstrated to induce high levels of circulating, polyfunctional T cell responses to survivin in clinical studies of advanced ovarian cancer patients, and some of these responses have been associated with an ability to mediate tumour regression. Preclinical and clinical data have supported combining this T cell targeting treatment with other immuno-oncology agents, such as checkpoint inhibitors.
Epitope discovery is a crucial element in the development of vaccine candidates and drug therapeutics. In the Immune-oncology space, identifying neoepitopes and tumor-associated antigens provide new targets for cancer diagnostics and enable the tracking of patient responses to treatment. Whereas, monitoring T cell responses to gene therapy vectors and transgenes is important in evaluating efficacy and safety of the treatment. ProImmune provides industry-leading tools for antigen characterization, epitope mapping and immune monitoring. Novel epitopes can be identified by the highly sensitive ProPresent® Antigen Presentation assay, which utilizes LC/MS/MS to determine peptides that are naturally processed and presented. Additionally, the REVEAL® HLA-peptide binding assay eliminates the need for precious patient samples and allows for the screening of multiple proteins for potential T cell epitopes in a matter of weeks. Once the epitopes have been identified, they act as important markers for tracking treatment effectiveness in clinical immune monitoring programs. IFN-γ ELISpots serve as a robust measurement of antigen specific immune responses following patient vaccination and/or treatment, while antigen-specific CD4+ T and CD8+ T cell responses can be enumerated by the ProT2® MHC-Tetramers and Pro5®MHC-Pentamers, respectively. In this presentation, case studies will be shared that detail how ProImmune’s integrated platform has identified novel epitopes in the immune-oncology and gene therapy field and enabled the detection and measurement of functional T cell responses against those epitopes in the clinic. Emilee Knowlton gained her PhD in Infectious Diseases and Microbiology from the University of Pittsburgh under the direction of Prof. Charles Rinaldo, identifying immune responses to lytic infection with Human Herpes Virus-8. She joined ProImmune in 2013 after completing her Post Doc in Rinaldo’s lab. She works on the ProImmune team providing innovative solutions for clients that radically improve our understanding of both desired and unwanted immune responses.
CAR-T cells are an engineered T cell therapeutic in which a chimeric antigen receptor (CAR) (a foreign protein) is expressed on the surface of autologous T cells. This foreign CAR protein has the potential to elicit immune responses from the host immune system. The immune response could be (a) humoral, resulting in the formation of Anti-CAR antibodies and/or (b) cellular, resulting in the development of cytotoxic T cells that are specific to the CAR-T cell. A host vs CAR-T response may result in (a) the neutralization of the CAR, rendering the CAR-T therapeutic ineffective (b) or result in the CAR-T cells being killed by a cytotoxic T cell response akin to a host vs graft response. This may manifest as lack of persistence of CAR-Ts and as loss of efficacy. There may be other reasons for loss of efficacy, and hence understanding if immunogenicity is the reason for loss of efficacy will be key in determining whether a second dose may be meaningful, the timing of this second dose, the impact of lymphodepletion on immunogenicity etc.
The immune response to protein-therapeutics (immunogenicity) is an important safety and efficacy concern during drug development and regulation. Non-clinical assays that can be used in the early stages of clinical development and to identify at-risk individuals and sub-populations in the clinic are an important unmet need. The so-called MHC Associated Peptide Proteomic (MAPPs) assay directly identifies peptides derived from a protein of interest on a donor’s MHC-II proteins. Here we have applied this technique to address several questions related to the use Factor VIII (FVIII) replacement therapy, in the treatment of hemophilia A. Over a dozen FVIII products are marketed but most fall into 3 categories: (i) Purified from human plasma (PD-FVIII). (ii) Full-length FVIII manufactured using recombinant DNA technology (FL-rFVIII). (iii) A truncated, beta-domain deleted rFVIII (BDD-rFVIII). We investigated whether there are differences in the FVIII peptides found on the MHC-II proteins of the same individual when incubated with products from the 3 classes. Our principal findings are as follows: The number of unique FVIII peptides isolated, average length of peptides and range of peptide length were comparable for MHC proteins immunoprecipitated from cells from hemophilia A patients and healthy donors. When cells from the same donor was exposed to FL-rFVIII in the presence and absence of PD-VWF, fewer peptides were recovered in the absence of PD-VWF; FVIII peptides not recovered in the presence of VWF map regions of VWF-FVIII interaction. When dendritic cells from the same donor were incubated with FL-rFVIII or PD-FVII (both in the presence of PD-VWF); fewer FVIII peptides were recovered from the PD-FVIII. The peptides not recovered in the PD-FVIII did not map to the locations of FVIII-VWF interactions. For each donor, FVIII peptides identified by the MAPPs assay exhibited affinities for that donor’s MHC variants which was orders of magnitude higher than the affinities of all overlapping peptides from FVIII and VWF, or when compared to a million random peptides obtained from the human proteome. Zuben Sauna is a Principal Investigator and a Reviewer at the US Food and Drug Administration. His research interests lie in understanding the pharmacogenetic basis of the immune response to proteins used in therapeutic interventions as these affect efficacy and safety. His laboratory exploits a combination of computational, in vitro and ex vivo approaches to understand why some individuals and/or sub-populations develop immune responses while others do not. His work has published extensively in high impact journals such as Nature Biotechnology, Nature Medicine, Science, Science Translational Medicine and Nature Reviews Genetics. He received his Ph.D. from Poona University, India with subsequent training at the National Cancer Institute, Bethesda, USA.
The commensal microbiota profoundly shapes the immune response, both at mucosal surfaces and systemically. Alterations in the diversity and the composition of commensal intestinal microbiota have been reported in several autoimmune diseases. This talk reviews some of the innate and adaptive mechanisms engaged by the microbiota to affect the development of autoimmune inflammatory arthritis.
We are in an unprecedented time in our ability to manipulate the immune system for the antithetical outcomes of immune activation to treat cancer and immune tolerance to treat autoimmune disease and allergy. Thus, we have witnessed unprecedented responses to late stage and widely metastatic cancer via immunotherapeutic approaches and are increasingly exploring novel and highly targeted approaches to autoimmune and allergic diseases. Important cautionary tales pertaining to unexpected adverse effects arising from these clinical studies, and caveats for future studies will be discussed.
Immunogenicity is one of the most complex issues to address in drug design and development. I will provide an overview of the best tools for immunogenicity risk mitigation, including Mass Spectrometry antigen presentation assays, Dendritic cell-T cell assays to measure responses to fully formulated biologics, HLA-peptide Binding Assays, and naïve T cell Proliferation Assays to characterize individual epitopes. I will also discuss how the potential risk of first infusion reactions can be mitigated using whole-blood cytokine release assays. Jeremy Fry gained his DPhil. from the University of Oxford working with Prof. Kathryn Wood and Prof. Peter Morris developing gene therapy strategies to induce immunological tolerance in transplant recipients. Jeremy joined ProImmune in the R&D team to generate a new class of MHC multimer staining reagents. Since 2002, as ProImmune's Director of Sales, he has led the sales team in a growing business, focusing on technologies that radically improve our understanding of immune responses.
Immunogenicity of biologics in the clinic has the potential to cause severer sequela and alter or neutralize efficacy resulting in exposing patients to risk and terminated development programs. Pre-clinical risk assessment utilizing orthogonal methods has made it possible to prioritize drug candidates with a decreased immunogenicity risk or utilize protein engineering o reduce immunogenicity risk. Here we will present the immunogenicity tool box utilized at Bristol Myers Squibb to pre-clinically minimize immunogenicity risk. Jochem Gokemeijer has been working at Bristol Myers Squibb for 15 years in Immunogenicity and biologics drug discovery. Before that he was involved in two alternative scaffold biotech companies Phylos and Adnexus Therapeutics. Jochem did his undergraduate work in the Netherlands and graduate work at the Dana Farber Cancer Institute.
The introduction of therapeutic protein drugs is recognized as a major step forward in the management of many severe diseases in multiple areas. However, their optimal use is hindered by the development of unwanted immunogenicity, which occurs in up to 40% of treated patients. Therapeutic protein-induced anti-drug antibodies can alter drug pharmacokinetics and pharmacodynamics leading to impaired efficacy, loss of clinical response and serious safety issues in the case of cross- reactivity with endogenous proteins with vital functions. Numerous factors related to patients, products and administration are thought to contribute to therapeutic protein immunogenicity. Hence, there has been a growing interest over the past decade in developing methods to assess the risk of unwanted immunogenicity early during drug development and mitigate the risk when products reach the clinic. Here, I will discuss the various strategies and approaches to therapeutic proteins in silico, in vitro and in vivo immunogenicity risk assessment. Sophie Tourdot received her Ph.D. in T cell vaccine immunology at the University of Paris 5. Following her postdoctoral trainings at Imperial College London, she worked at the Pasteur Institute on HIV vaccine design then progressed to Stallergenes-Greer and ITS, where she lead the Pre-clinical immuno-pharmacology teams. She then worked at the French National Research Institute for Health and Medical Research (INSERM) as the Scientific Project Manager of the IMI-funded ABIRISK project, a consortium of 38 partners from the pharmaceutical industry and academic research centers across Europe, Israel and the United-States. The ABIRISK program focused on the analysis of underlying biological mechanisms, clinical relevance and prediction of unwanted immunogenicity of biopharmaceuticals. In her current position at Pfizer in the BioMedicine Design department, Sophie has responsibilities for biologics immunogenicity risk assessment and mitigation. She is also the European Immunogenicity Platform Scientific Affairs Director.
Despite the significant growth in number and diversity of biotherapeutics over the past twenty years and the overall clinical success, immunogenicity (IG) prediction remains elusive and has shortcomings. Indeed, the production of anti-drug antibodies may alter drug pharmacokinetics and pharmacodynamics, efficacy, and/or cause adverse events or have no consequences at all. The assessment of IG in the clinic is a regulatory requirement, yet efforts are usually in place at the pre-clinical phase to assess IG risk for guiding product development. The adoption of in-silico tools to identify regions of the drug that might trigger a T- or a B-cell response has gained momentum due to their high-throughput capabilities and the minimal cost of operation per analyzed sequence. However, the theoretical underpinning of the most widely adopted epitope mapping platforms, particularly for T-cells, is still evolving with the most recent data concerning the biology, biochemistry and biophysics of antigen processing and presentation. An evaluation of these experimental observations will be presented, such as the flexible nature of peptide binding to MHC molecules, the effect of proteolysis on epitope generation, the role played by auxiliary molecules in peptide selection, and the use of peptide affinity as a proxy for immunodominance. Finally, a strategic path on how to integrate these datasets into the in-silico prediction of IG will be proposed. Andrea Ferrante attained his M.D. at the University of Genoa (Italy) School of Medicine in 2003 and his M.B.A. in 2017 at the University of Alaska Fairbanks. His postdoctoral training includes 3 years of residency in Clinical Pathology (Italy) and 7 years of research at the Blood Research Institute, Milwaukee (WI). In April 2012 he joined the faculty at the University of Alaska Fairbanks as an Assistant Professor in the Department of Biology and Wildlife and the Institute of Arctic Biology. His research activity has focused primarily on the molecular basis of antigen processing and presentation by MHC class II molecules. He has been the recipient of several grants as PI and co-PI, including a 5-year NIH R01 award and a 3-year Aspire Pfizer Award, and has authored about 20 publications including original research, review articles and conference proceedings. He joined Lilly in May 2017 to support the effort in developing an integrated group aimed at assessing and mitigating immunogenicity risk of biotherapeutics.
Rodd Polsky is an Investigator responsible for immunogenicity assay development within the Bioanalysis, Immunogenicity and Biomarkers organization at GlaxoSmithKline. Rodd received his Bachelor’s degree in Biology from Drexel University. Since that time, Rodd has spent the past 25 years providing preclinical and clinical immunogenicity support, including assay and study design, development, validation, and study support for the detection of anti-drug antibodies against therapeutic proteins.
Dr. Janssen of Apitope described their antigen processing independent epitopes (apitopes), which were evaluated as antigen-specific immunotherapeutics for autoimmune disease. She demonstrated clinical efficacy of ATX-MS-1467 for MS treatment , as well as highlighted some early Phase I data for ATX-GD-59 for Grave's Disease.
Human T lymphotropic virus -1 (HTLV-1) is a retrovirus which causes an aggressive blood cancer, adult T cell leukaemia/lymphoma (ATL), in 5% of carriers. HTLV-1 preferentially infects CD4+ T cells, and the virus genome is found inserted into the DNA of malignant cells in ATL. We know that individuals who poorly control the virus have a particularly high risk of developing cancer, and that the viral burden is principally controlled by the cytotoxic T lymphocyte (CTL) response to HTLV-1. Here I will describe some novel approaches we have developed (1) to assess the efficiency of CTL killing of HTLV-1-infected and leukaemic T cells ex vivo and (2) to quantify and characterise clonally expanded HTLV-1-infected malignant T cells.
Dr. Paul Brenchley of Manchester University describes the progress in defining the key peptide epitopes in the target antigens of membranous nephropathy. He identifies HLA-DQ restricted 5 peptides from a recombinant PLA2R using the ProPresent assay.
Epitope identification has emerged as a crucial stepping stone to study immune responses. It aids the discovery of targets for the development of new therapeutics and vaccines but can also identify potential areas of a therapeutic that are likely to generate neutralising anti-drug antibodies resulting in failure of treatment. Crucially, as antigen-specific immune responses are being routinely monitored to investigate the efficacy of a given treatment, thorough epitope mapping has dramatically enhanced the efficiency of these studies by flagging relevant epitopes to use for immune-monitoring, optimising the use of researchers time and resources and saving precious patient samples. Recently, we have contributed to confirming the success of ParvOryx, the first phase I/IIa clinical trial employing oncolytic rodent protoparvorirus to treat patients with recurrent glioblastoma. Using our mass-spectrometry-based ProPresent® Antigen Presentation assay on parvovirus-infected glioma cell lines, we have identified a panel of epitopes that are derived from both glioma cancer cells and the parvovirus. This has subsequently allowed the monitoring of antigen-specific immune responses by functional IFNγ ELISPOT. Results have shown that the majority of patients mounted an antiviral-specific response, demonstrating responsiveness to the treatment. Furthermore, some patients have also shown a low yet significant response to glioma antigens which could partially account effectiveness of treatment and for the positive clinical outcome of the trial. In this talk, we will also be detailing how epitope identification and immune monitoring multimers are powerful tools to boost the advancements of other gene therapy approaches notably Adeno-associated viruses and HSV.
Dr. Yongliang Zhang describes the role for MAPK Phosphotase 5 (DUSP10) in inflammation.
Dr. Guy Hermans of Isogenica describes the benefits of synthetic antibody libraries to reduce synthesis error, avoid immunogenicity liabilities and yield broadly species crossreactive binders.
Dr. Ilaria Espositio describes the use of MHC Class II Tetramers to track HCV specific CD following adminisration of their ChAd vaccine. She outlines the phenotype, funcitonal capacity and proliverative capacity of the HCV-specific cells.
Dr. Isabelle Turbica describes a DC Activation assay to evaluate surface activation markers, mRNA expression, cytokine release and cell signaling in response to native and aggregated antibodies
Dr. Eleni Chantzoura describes how pentamers and the Agenus TCR display platform was used to identfy phosphopeptide-specific TCRs.
Dr. Joleen White of EMD Serono provides case study data on Avelumab, an anti-PD-L1 antibody and details the safety assessment from Phase I/Ib and Phase II studies.
The last few years have shown a strong paradigm shift in cancer therapies with the approval of antibodies targeting immune checkpoints (CTLA-4, PD-1 and PD-L1). These immune checkpoint blockers (ICBs) are associated with a strong and long-lasting response in patients suffering from different malignancies. However, there is still a high proportion of patients showing intrinsic or acquired resistance to ICBs. Although the mechanisms associated with the lack of immune response are multiple, the presence of highly anti-inflammatory regulatory T cells (TRegs) in the tumour microenvironment (TME) is known to negatively affect the response to these therapies. Similarly, high TReg levels in the TME have been associated with poor prognosis in several cancers. Together, this highlights the potential of targeting and depleting TRegs to enhance anti-tumour responses. The Inducible T-cell costimulator (ICOS/CD278) is related to the CD28 superfamily and is induced when T cells get activated. ICOS expression levels vary in different immune cell subtypes and in different tissues. In preclinical mouse tumour models, TRegs (CD4+/FOXP3+) constitutively express ICOS on their surface and the expression of ICOS on TRegs is significantly higher than that on effector T cells (TEffs). In addition, ICOS expression on TRegs is higher in the TME than in the blood or spleen, which makes it a strong candidate for preferential depletion of TRegs in tumours. By immunizing Kymice™ in which the endogenous Icos gene has been knocked out, we identified a novel, fully human antibody called KY1044 that cross-reacts with mouse ICOS. KY1044 is an anti-ICOS subclass G1 kappa monoclonal antibody that selectively binds to dimeric ICOS (Fc fusion) with an affinity of less than 2nM. Using in vitro and in vivo approaches we demonstrate that KY1044 has a dual mechanism of action: (1) it promotes the preferential depletion of intratumoural ICOShigh TRegs resulting in an increase in the TEff:TReg ratio in the TME; and (2) it stimulates ICOSLow TEff cells. Using the mouse effector enabled version of KY1044 (mIgG2a) we confirm, using syngeneic models, a strong anti-tumour efficacy as monotherapy or in combination with surrogates of ICBs. We also demonstrated a tumour antigen specific immunity, as highlighted by the rejection of the original tumour cells in animals cured of the disease and re-challenged by the same cell line. Noteworthy, Pharmacodynamic studies demonstrate long-term depletion of TRegs and a significant increase in the TEff:TReg ratio in response to KY1044. In summary, our data demonstrate that targeting ICOS with KY1044 is a valid approach for manipulating the immune system and for inducing a strong anti-tumour response in several indications. The data presented here also warrant the assessment of KY1044 in cancer patients in a clinical trial.
While attempting to generate tumour specific T cell clones from leukaemia patients, we isolated a CD8+ T cell clone (6C5) with the capacity to recognize a t-butyl modified peptide (LLSY(3-tBu)FGTPT) presented by HLA-A*0201. This unusual and unnatural epitope is a poor binder to HLA-A*0201 and was likely created as a by-product of FMOC peptide synthesis. Since this epitope does not occur in nature, we sought to understand how the structural features of the LLSY(3-tBu)FGTPT/HLA-A*0201 complex could contribute to immunogenicity. We demonstrated that 6C5 T cells were specific for the type of chemical modification on peptides, and by using peptide libraries showed that 6C5 recognized several peptides derived from self-proteins. These peptides were dissimilar in sequence to LLSY(3-tBu)FGTPT but were more potent at inducing T cell function. Structural studies of three different peptide/HLA-A*0201 complexes are ongoing to determine how peptide structure can contribute to this cross-reactivity. Overall our studies show that chemical modification of peptides can influence immunogenicity but can also generate T cells with unexpected specificities.
T cell immunity directed against tumor encoded amino acid substitutions has been reported in humans with cancer, thus implicating missense mutations as a source of patient-specific neoantigens. Emerging data suggest that T cell responses directed at these private neoantigens, presented in the context of Human Leukocyte Antigen (HLA) cell surface molecules, are a major factor in the clinical activity of checkpoint inhibitors and adoptive T cell therapies. Recently, we demonstrated that vaccination against tumor missense mutations increases the antigenic breadth and clonal diversity of neoantigen-specific T cell immunity in patients with metastatic melanoma. In this presentation, I will discuss our pipeline for neoantigen identification and vaccination approach. Additionally, the effect of tumor heterogeneity in the neoantigen landscape as well as the characteristics of neoantigen T cell immunity elicited by vaccine will be presented.
The gastrointestinal (GI) tract is home to trillions of commensal bacteria that play an important role in nutrition, immune system development and host defence. In inflammatory bowel disease (IBD), a chronic debilitating disease of the gastrointestinal tract, there is a breakdown in the healthy dialogue between our body and our microbial residents resulting in chronic immune attack in the bowel. In this presentation I will review key host and microbial pathways that maintain intestinal homeostasis and discuss how understanding these pathways may provide new therapies for the treatment of chronic inflammatory diseases.
Valerie Quarmby is a Staff Scientist in the Department of BioAnalytical Sciences at Genentech. At Genentech, Dr. Quarmby has developed bioanalytical methods & strategies to enable IND, BLA and related filings for: Nutropin, Xolair, Raptiva, Rituxan, Avastin, Lucentis, Perjeta, Kadcyla and Tecentriq. She has also developed bioanalytical methods, platforms and strategies for many of the therapeutic proteins in the gRED drug development pipeline. Valerie also consults on bioanalytical and related strategies across the Roche group. She is the past Chair of the AAPS Therapeutic Protein Immunogenicity Focus Group and a member of the 2010-2015 USP Immunogenicity Testing Expert Panel. Valerie is the co-author of several AAPS sponsored industry guidance documents and many publications in peer-reviewed journals. Valerie Quarmby holds a Ph.D. in Hormone Physiology from the Imperial Cancer Research Fund and the University of London in England. Dr. Quarmby was a Postdoctoral Visiting Fellow at the US National Institutes of Health, and then joined the Laboratories for Reproductive Biology and the Department of Pediatric Endocrinology at UNC-Chapel Hill. Prior to joining Genentech, Dr. Quarmby worked in the field of clinical diagnostics at Bio-Rad Laboratories and at Endocrine Sciences/Esoterix. In 2014, in recognition of her many contributions to the pharmaceutical industry, Dr. Quarmby was awarded AAPS Fellow Status.
The field of Immunogenicity Risk Assessment has blossomed of late in the pharmaceutical industry. In silico and in vitro analysis for immunogenicity risk is now a standard practice at Bristol Myers Squibb. Here, we describe the various tools that we use and detail case studies where these tools were used to identify lead candidates or help to deimmunize lead candidates when significant risk was identified.
Erik Meyer’s formal education included undergraduate studies at the University of Texas in Austin, graduate studies at Massachusetts Institute of Technology, and post-doctoral work at Texas A&M. I then joined a biotech start up, Xenomics isolating urine DNA for diagnostic analysis. At Huntingdon Life Sciences and Tandem Laboratories, I learned the nuances of Good Laboratory Practices and immune assay development for PK and immunogenicity detection, which have been useful for supporting anti-drug antibody detection for various clinical projects at GSK.
Immunogenicity (development of anti-drug antibodies) is a significant impediment to development and licensure of any therapeutic-protein. Recent progress in the development and use of non-clinical and pre-clinical assessments of immunogenicity will be presented along with examples demonstrating good predictive outcomes. I will illustrate how judicious application of these tools can permit better decision making during drug-development, licensure, and clinical-trials.
Head of R&D and Scientific Founder of Déclion Holdings LLC, has been interested in peptide vaccines and immunomodulators for the last 25 years. His previous positions include Head of R&D at Peptimmune Inc., Senior Scientist at Praecis Pharmaceuticals and Cardion AG, and Senior Investigator at Leiden University Medical Center. He completed his postdoctoral training at the Mayo Clinic in Rochester MN in the laboratory of Dr. Chella David, a pioneer in the field of Immunogenetics.
Though deep sequencing of T-cell receptor (TCR) repertoires can now be performed routinely to reveal the scope of repertoire diversity, these TCR-seq experiments do not provide any information regarding the antigenic targets of individual TCR clonotypes nor the scope of reactivity encoded within the TCR repertoire. Functional testing of T-cell receptors against large sets of possible antigens is required in order to link TCRs-of-interest discovered in TCR-seq to their cognate antigens. However, existing methods for T-cell antigen identification are limited in scalability and, therefore, inadequate for use in large-scale epitope discovery efforts that are necessary to decode the complexity of T-cell reactivity. We have developed a novel high-throughput screening approach for T-cell antigen discovery that has been validated in model mouse systems to match the specificity and sensitivity of current methods but with the added advantage of being able to screen greater than 100x more antigens than is currently practical with existing strategies. This approach employs a granzyme-B (GZMB)-sensitive fluorescent reporter gene linked to libraries of short peptide-encoding “minigene” sequences. Minigene-reporter cassettes are lentivirally transferred into a pool of sero-matched antigen presenting cells (APC), which can then be co-incubated with T-cell lines of interest. Detection of the reporter signal in targeted cells allows for the isolation of APCs carrying putatively antigenic minigenes. These minigenes are recovered by PCR and characterized by deep amplicon sequencing to reveal the landscape of antigens that are recognized by the input T-cells. Currently, we are working towards transitioning the approach to human cells and also leveraging its high-throughput capacity to evaluate the cross-reactivity of murine and human model TCRs against large libraries of randomized synthetic minigenes. The objective of these studies will be to explore the size and characteristics of the peptide repertoire that individual TCRs can recognize from a non-endogenous set of antigens, which, in addition to being a widely discussed but largely unexplored area of T-cell biology, is an important consideration for the development of safer, more precise immunotherapies and biopharmaceuticals.
Allergen cross-reactivity is based on shared IgE-binding which is due to shared sequence and higher level structure (charge and shape). Protein allergens can be uniquely related by cross-reactivity and specific IgE binding epitopes can be important in predicting cross-reactivity potential. Refining the measures of sequence similarity may be used to establish criteria that identify potential epitope homology among groups of allergens. Selected allergens were examined using IgE binding epitope sequences and these were used to determine how the FASTA algorithm could be used to identify a threshold of similarity. The allergens peanut Ara h 1 and Ara h 2, shrimp tropomyosin Pen a 1, and Birch tree pollen allergen, Bet v 1 were sources of known epitopes. Each epitope or set of epitopes was inserted into random amino acid sequence to create hypothetical proteins used as queries to an allergen database. A statistical measure of sequence similarity (E-value) was used to identify a threshold. Hypothetical proteins were examined for alignments with allergens and each was noted for the ability to match the epitope’s source allergen as well as match any cross-reactive or other homologous allergens. A minimum FASTA E-value range was identified that could identify the presence of homologous allergens and which could be used to screen novel proteins for cross-reactivity potential.
Bernard Maillère is currently director of research and head of the laboratory of immunochemistry of the cellular immune response at Commissariat à l'Energie Atomique (CEA), a French Governmental Research organism. His works mainly deal with the prediction of immunogenicity and identification of T cell epitopes in humans. Bernard is currently workpackage leader of the European IMI project ABIRISK dedicated to immunogenicity of therapeutic proteins
Immune complexes modulate immune highways and DC trafficking Antibodies are critical for defence against infection but may also be pathogenic in some autoimmune diseases. Many effector functions of antibody are mediated by Fcγ receptors (FcγRs), which are found on most immune cells, including dendritic cells (DCs). We demonstrate that FcγR engagement by IgG immune complexes (IC) stimulates DC migration from peripheral tissues to the paracortex of draining lymph nodes. Using intravital two-photon microscopy, we observed that local administration of model IC or autoantibody-containing IC from patients with systemic lupus erythematosus (SLE), resulted in dermal DC mobilisation. We confirmed that dermal DC migration to lymph nodes was CCR7-dependent and increased in the absence of the inhibitory receptor, FcγRIIb. Together, these data suggest an additional mechanism by which ICs might drive autoimmunity in SLE via the inappropriate localisation of autoantigen-bearing DC and may also have implications for boost strategies in vaccination
A large number of different classes of tumour-associated antigens provide specific targets for immune therapy of cancer. These include antigens expressed by oncogenic viral vectors such as Human Papilloma Virus (HPV), as well as a whole array of mutated oncogenes, abnormally glycosylated proteins and carcino-embryonic gene products that are expressed at elevated levels in different malignancies. In addition to these common antigenic targets, recent data has demonstrated the presence of other, entirely patient and tumour specific mutations. Both the common and the private tumour-associated antigens provide potential targets for the immune mediated eradication of cancer. Such immune therapy strategies are of particular relevance to the eradication of the residual cancer cells, which can contribute to possible relapse and recurrence, despite a successful initial response to therapy. The most promising of the new immune therapy based approaches include the use of antibody based blockade of immune inhibitory check points (e.g. anti-CTLA4, anti-PD1/PDL1, etc.). The inhibition of these feedback loops allows the stimulation and expansion of specific populations of tumour specific T cells. An alternative and highly promising new form of immune therapy is the generation of autologous and allogeneic T cells expressing antigen specific T cell receptors, including chimeric antigen receptors (CAR T-cells), that are able to recognise and lyse tumour cells. Other strategies showing moderate clinical efficacy include the use of autologous dendritic cells that are pulsed with tumour-associated RNA, proteins and peptides, or with whole tumour lysate. Alternatively, autologous cancer cells can themselves be genetically modified to serve as whole cell vaccines. Yet other exciting new developments include the identification of new adjuvants and vaccination strategies for cancer specific induction of protective and therapeutic cellular immunity. The combination of such new vaccination strategies, combined with identification of individualised cancer specific mutations (e.g. by exome sequencing, RNAseq, whole genome sequencing, etc.) is holding out the promise of substantially more effective vaccination strategies. Given the rapid pace of these developments, the treatment of many cancers is now poised for dramatic improvements
SOBI is developing novel biopharmaceutical drugs based on the antibody-like Affibody scaffold. The molecules are fusion proteins composed of target specific Affibody domains of non-human origin, fused to the human IgG1-Fc for half-life extension. The E. coli produced non-glycosylated Fc fusions are devoid of Fc-gamma receptor and C1q mediated effector functions but still retain FcRn-mediated recycling with associated decreased clearance. In the screening process to select the most promising drug candidates, a multi-tiered approach was used to evaluate immunogenicity of Affibody-Fc fusion proteins. Several in silico platforms were used for a broad primary screening. Next we investigated which peptides were presented on MHC-II after protein uptake and processing by dendritic cells (DCs). Predicted or presented peptides, protein domains or full-length proteins were next used to stimulate CD4+ T-cells using PBMCs or DC:T assays. The selected candidates had a DC:T assay response-index similar to therapeutic grade human antibodies or human Fc indicating low immunogenicity
An immunogenicity risk assessment incorporates risk factors related to patients, products and the way the product is given to the patient. Prediction of the clinical impact of immunogenicity could be possible through a quantitative understanding of these risk factors and how the immune system operates. In vitro assays contribute to this quantitative understanding of risk and can be used during the discovery phase to inform risk mitigation strategies such as selection of lower risk leads.
It is now well established that many tumors are under the surveillance of the host immune system and that anti-tumor adaptive responses are critical for the clinical outcome of patients. One consequence of this paradigm is that monoclonal antibody (mAb) therapy that had been viewed for a long time only as acting through the involvement of innate immunity is now thought to be efficient due to its ability to trigger or reinforce adaptive anti-tumor responses. Notably, reports based on in vivo preclinical models using CD20+ or HER2/neu+ tumor-bearing mice have suggested that anti-CD20 or anti-HER2/neu mAb treatments lead to long-term survival through the induction of adaptive T-cell anti-tumor responses, considered as a “vaccine effect”. 1,2,3,4 Thus, the aim of the present study was to monitor changes in adaptive immune T-cell compartments in peripheral blood from Follicular Lymphoma (FL) patients during anti-CD20 (rituximab, RTX) treatment using multi-parametric phenotyping, transcriptomic and functional analyses. High-grade FL patients (n=30) were enrolled in the study. Blood samples were obtained before treatment (T0), during RTX infusion combined to CHOP chemotherapy regimen (T-R-CHOP), and during RTX maintenance therapy (T-Rm). Multi-parametric flow cytometry analysis underlined profound differences in naive, regulatory and memory T-cell compartments between patients at diagnosis (before therapy) and healthy donors. Furthermore, inhibitory immune checkpoints expressing-T cells were much more frequent in the blood of patients compared to healthy donors. Hierarchical clustering of patients based on the integrative analysis of flow cytometry data showed differential activation status of peripheral T cells among individuals prior to any treatment. Follow-up of blood T-cell compartments by flow cytometry during treatment showed an overall decrease in the percentage of regulatory T cells and T cells expressing inhibitory checkpoints. Several clusters of patients could be defined, according to different parameters analyzed throughout the treatment. Transcriptomic analysis of immune-related genes in 16 patients revealed that 174 genes were significantly differentially expressed between T0 and T-RCHOP, 68 between T-R-CHOP and T-Rm, and 61 between T-Rm and T0, indicating that the major changes occurred during initiation treatment. The frequencies of individuals exhibiting T-cell responses against CMV/Flu/EBV peptides or immunogenic CD20 peptides (defined by in silico, in vitro and in vivo approaches) were similar between patients and healthy donors. However, the intensity of specific T-cell responses was lower in all the patients tested, although these responses appear to be heterogeneous. Overall, T-cell compartments are profoundly affected in patients with high-grade FL prior any treatment. Our data indicate that R-CHOP followed by RTXm modulates T-cell activation status and function in a heterogeneous way among FL patients. The hierarchical clustering of patients based on the phenotypic and functional analysis of T-cell compartments pave the way for the definition of new immune biomarkers correlating with the clinical outcome of patients.
Display of foreign surface antigens on baculovirus as pseudotyped viruses for immunological studies and applicationsBaculovirus is an insect virus, and has long been used as a safe microbial agent for high level vaccine production and gene delivery into mammalian cells with high efficiency. Baculovirus has a major surface protein named GP64, which is a trimeric protein that functions for receptor recognition allowing baculovirus to enter the host cells. We found that the domain underneath this protein called cytosolic terminal domain (CTD) is crucial for the anchoring of any membrane protein targeting to the envelope of the baculovirus. By adding the CTD of GP64 to the spike protein of SARS virus, we have successfully anchored this protein onto baculovirus, and this functional pseudovirus allowed us to prove that spike protein is responsible for the induction of cytokine storm, and in turn induces illness of the patients. In further experiments, we found that this technology can be applied to display the surface proteins of influenza viruses. We have fused CTD from GP64 to the C-terminal of hemagglutinin, a trimeric major surface protein of influenza virus, and generated baculovirus-based pseudovirus. This pseudovirus was used to inject mice for antibody production. After three antigen shots, the mouse was sacrificed and analyzed the generated monoclonal antibodies. We found both neutralizing and non-neutralizing antibodies were induced. We also presented the neuraminidase (NA) protein of the flu virus onto the envelope of baculovirus and showed that NA, a tetrameric protein, can be functional on baculovirus for enzymatic assays. Therefore, surface display of foreign proteins on baculovirus with the assists of GP64 CTD is a useful technology to apply or study those major surface antigens of detrimental viruses
Tim Elliott left the University of Oxford with a first in Biochemistry in 1983 and completed his PhD in cancer immunotherapy at the University of Southampton in 1986. He did his postdoctoral training at MIT with Herman Eisen at the Center for Cancer Research. In 1990 he returned to the University of Oxford to join the Institute for Molecular Medicine as a Wellcome Trust Research Fellow, joining a key group of immunologists studying antigen presentation at the molecular level: where he continues to be a world leader with over 130 research articles on the subject. In 1993 he was appointed to a lectureship and later a Professorship at Balliol College, University of Oxford, as a Wellcome Trust Senior Fellow in Basic Biomedical Science. In 2000, he moved to the University of Southampton as Professor of Experimental Oncology and five years later became Associate Dean for the Faculty of Medicine. In 2015 he stepped down from this role to take up Directorship of the new Southampton Centre for Cancer Immunology which will open in 2017. He is a Deputy Director of the interdisciplinary Southampton Institute for Life Sciences and a Fellow of the Academy of Medical Sciences. He has incorporated discoveries in the areas of antigen processing, T cell regulation and immunodominance into the development of new cancer immunotherapies and is the recipient of a Royal Society/Wolfson Research Merit Award
In cancer cells, the disruption of normal biochemical signaling results in aberrant post-translational modifications, including protein phosphorylation. These mis-phosphorylated proteins can be processed into antigenic peptides by the cellular machinery, resulting in presentation of this novel type of antigen on MHC molecules. PhosphoSynVax is Agenus’s third heat-shock protein-based vaccine platform. PhosphoSynVax is designed to induce immunity against this novel class of phospho-neo-epitopes specific to malignant cells of multiple donors in various cancer types. Based on mass-spectrometry and bio-informatic approaches, we have a library of proprietary phosphopeptide tumor target antigens from different cancers, including lung cancer, specific leukemias, ovarian cancer, colon cancer and others. This approach may enhance immune system recognition of phospho-neo-epitopes, leading to the destruction of cancer cells in various patients across cancer types.
Christine Falk graduated in Biology at the Ludwig-Maximilians-University in Munich and performed her PhD thesis at the Institute for Immunology at LMU Munich (director G. Riethmüller) in the laboratory of D.J. Schendel on autoimmunity and the regulation of NK-like T cells and NK cells. As a postdoctoral fellow at the new Institute of Molecular Immunology (director D.J. Schendel) at the Helmholtz Center for Environment and Health (GMGU), she studied recognition of virus-infected cells and tumor cells by T and NK cells. As senior postdoctoral fellow and head of the research group “Immune Monitoring” at the German Cancer Research Center DKFZ in Heidelberg from 2006 until 2010, she was working on the microenviroment of solid tumors and its impact on tumor recognition by T and NK cells. In her current position since 2010, she is head of the Institute of Transplant Immunology, IFB-Tx, at Hannover Medical School MHH where she has established an immune monitoring laboratory investigating adaptive and innate immune responses in the context of stem cell as well as solid organ transplantation, in particular lung, heart, liver and kidney transplantation. The addition of transplant immunology to her former field of tumor immunology is reflected by focusing on common immunological mechanisms for rejection of both tumors and allografts that may open new perspectives for immunological interventions to achieve tumor rejection on one hand and on the other hand, immunological tolerance, the ultimate goal of solid organ transplantation
Miles Carroll joined Public Health England as Deputy Director, Head of Research at Porton Down in September 2008. In his current role he is responsible for greater than 250 scientists and support services personnel. He also has strategic and operational control to ensure that the Department is at the forefront of translational research in the areas of emerging diseases, diagnostics and decontamination, host pathogen interactions, infectious disease vaccines and therapeutics. Miles gained his PhD from the Medical Faculty at the University of Manchester which enabled him to obtain an International Fogarty Fellowship and continue his studies on recombinant poxviruses at the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA. On his return to the UK, Miles joined Oxford Biomedica as Vice President of Immunotherapy. At OBM Miles invented the cancer vaccine, TroVax and led the pre-clinical and Phase II development programme. Miles has authored greater than 60 publications primarily in the field of recombinant vaccines and emerging diseases, and has more than 10 granted patents. He has acted as an advisor to several biotech companies, appeared as an expert witness in both European and US patent cases. He also serves on the Scientific Review Boards of both Animal and Plant Health Institute (Weybridge) and Defence Science and Technology Laboratories. Miles is honorary Professor of Vaccinology at the Medical Faculty of the University of Southampton.
Dr. Herman Waldmann reviews the mechanisms of tolerance induction and discusses strategies to evoke tolerance to immunogenic antibodies.
Dr. Rarnaz Fallah Arani reviews the challenges how to translate experimental findings into clinical applications. She also describes an invitro antigen-driven recall/restimulation assay and in vivo transplanation models to evaluate drug function and effects upon with rawl.
Dr. Bernd Schlereth of Covagen presents their bispecific Fynomer/FynomAb technology for treatment of inflammatory disease. He describes their PK and Immunogenicity testing strategy including multiple assays to asses early and late ADA response.
Dr. Bernard Maillere summarizes data from the Abrisk consortium. He shows that predicted epitopes from the T cell-based assays and mass spec antigen presenation assays weere consisten with epitopes detected in patients, while in silico analysis was overpredictive.
Dr. David Wraith, founder of Apitope, describes how peptide immunotherapies can induce antigen-specific desensitization. He describes how the REVEAL binding assay was used to identify pan-allele binding epitopes, which can servce as potential targets for tolerance induction.
Dr. Peter Adler Wurtzen of ALK discusses the immunological changes during allergy immunotherapy and their link to clinical effect. He demonstrates correlations to various immunological markers and changes in both antibody and T cell repetoires.
Dr. Paul Moss describes the CD8 and CD4 T cell immune responses to CMV infection. He describes the importance of Class II Tetramers to better understand the interplay of a chronic infection and immune control.
Dr. Wei Xu of the Roche Innovation Center discusses the use of combination immunotherapy to increase priming and activation, antigen presentation and recognition of cancer cells. He used an MHC Class I Pentamer to show cancer-specific immunity and expansion of CD8 T cells in response to DNA vaccine plus immunotherapy.
Studies over the past 20 years have clearly defined and extensively characterized Foxp3+ T regulatory cells (Tregs) as major players in the control of most aspects of immune responses as well as playing a potential role in non-immunologic sites (fat, muscle). We are now at a point where studies have been/or will be initiated in the clinic using cellular biotherapy with Tregs, augmentation of Treg numbers/function with IL-2, and modulation of Treg function with small molecules and antibodies. Yet, many factors involved in Treg biology remain poorly studied. I will focus this talk on three key issues: 1) control of Treg homeostasis by TCR and co-stimulatory signals; 2) the role of transcription factors in modifying certain Treg functions; and 3) a critical review of suppressor mechanisms used by polyclonal and antigen-specific Tregs.
As it is associated with a 500.000 deaths each year, breast cancer engenders a high burden. Rather than continue to add to the mounting costs of incidence and recurrence, we believe that we should reposition ourselves and advance promising, affordable, safe strategies for the primary and secondary prevention. Vaccination has been one of the most successful approaches to reduce mortality rates of infectious diseases and more recently cancer. The best approach is to target aberrantly expressed ‘self’ antigens that contribute to the pathogenesis. While targeting of self antigens implies breaking tolerance and causing autoimmunity, this is rarely observed. The reasons for the lack of autoimmunity are not clear, but according to our hypothesis, significant vaccine-induced responses are generated against subdominant epitopes, expressed only in the local tumor microenvironment, whereas no immune response is generated against immunodominant epitopes of healthy tissues. The result is protection in the absence of autoimmune sequelae. In addition, an attractive vaccine approach involves generation of immunity to several antigens covering multiple cancer cell types (epithelial tumor cells, breast cancer stem cells and stromal cells). To produce such a safe and effective vaccine, we’re aiming to achieve key milestones, namely identifying subdominant epitopes from several cancer associated antigens and identifying vector systems that can induce durable CD4 and CD8 T cell responses.
Accumulated evidence gathered in the last three decades demonstrated that some members of the Parvoviridae family, belonging in particular to the species Rodent protoparvovirus, have natural antineoplastic activity in cell culture and animal models, and that human tumor cells can be targets for this activity. In an immunocompetent rat glioma model, the H-1 parvovirus (H-1PV) was able to efficiently cure gliomas while raising an antitumor memory immune response. This oncosuppressive effect was observed after virus administration through intratumoral, intravenous and intranasal routes, and appeared to rely on both the direct oncolytic activity of H-1PV, and its handover to the host immune system. The parvovirus was also capable of inducing the regression of human glioma xenografts in immunodeficient rats. These studies have laid the foundations for the launch of a first phase I/IIa clinical trial in which H-1PV is currently undergoing evaluation for its safety and first signs of efficacy in patients with recurrent resectable glioblastoma multiforme progressing in spite of conventional therapies (ParvOryx01 trial). While no clinical conclusions, besides safety, can be drawn at this stage from the trial, there are intriguing laboratory measurements in resected tumors and patients’ blood. Immunohistochemical and FISH analyses provided evidence for virus efficiency in crossing the blood-brain barrier, spreading throughout the tumor and getting expressed in tumor tissues. The possible impact of these direct viral effects on the tumor fate has to be balanced against evidence of immune infiltration and vascular disruption in infected tumors, and of systemic immune responses in treated patients.
Predicting incidence of clinical immunogenicity is currently challenging, due to the intrinsic complexity of the immune system and the involvement of various risk factors. To help address the challenge, we have developed a mechanistic, multi-scale mathematical model, by recapitulating fundamental biological mechanisms. The key strength of this systems model lies in its capacity to integrate various risk factors, e.g., T- and B- epitopes, patients’ HLA background, naïve T and B cell numbers, into the underlying biology. The model can simulate the immune response at both individual and population level, and provides many clinical-relevant predictions, e.g., incidences for immunogenicity and loss of efficacy. An example of simulating human population immune responses against a therapeutic protein will be provided, to illustrate potential applications in drug development. The current model can be subjected to rigorous experimental validation, and could eventually serve as a tool to integrate various preclinical and clinical results for immunogenicity prediction and mitigation.
Stealth liposomes are promising nanomedicine tools for bringing cytotoxic drugs to their target. However, the release of a drug only at the target has remained elusive. We engineered a natural sensory ion channel into a stimulus-sensitive nanovalve and incorporated it into the stealth liposomes. In the absence of the stimulus, e.g. during circulation in the blood, the nanovalve stays closed. However, upon sensing a target-specific stimulus, it opens a large, non-selective aqueous pore on the surface of the liposome and releases the drug payload at once. Currently, the nanovalve detects the pH differences in the physiological pH range with 0.2 pH unit precision in vivo and the wavelength of the light. It can be engineered to detect other signals. Even though such triggered drug delivery system is very advantageous, the bacterial origin of the ion channel poses a risk of immunogenicity. We addressed this point by producing alternative nanovalves from the homologues ion channels from different organisms. After predicting the potential immunogenicity of these ion channels in silico, the relative immunogenicity of our liposomal formulations with individual ion channels were tested by Dendritic cell-T cell assay from ProImmune. We obtained very promising results, which will be discussed in the meeting..
The regulation of immune responses to self and foreign antigens is vitally important to limit immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune and allergic conditions can be reinforced by tolerance induction with peptide epitopes; this presentation will focus on the mechanisms involved. Peptides must mimic naturally processed epitopes. Peptide induced peripheral tolerance is characterised by the generation of anergic, IL-10 secreting CD4+ T-cells with regulatory function. CD4+ T-cells become anergic following their first encounter with peptide. The loss of proliferative capacity correlates with a cytokine switch from a pro-inflammatory to a phenotype characterised by secretion of the anti-inflammatory cytokine IL-10. IL-10 Treg cells suppress dendritic cell maturation, prevent Th cell differentiation and create a negative feedback loop for Th driven immune pathology. Tolerance induction involves upregulation of transcription factors controlling IL-10 and inhibitory receptors limiting T cell signalling. Results from clinical trials of peptide immunotherapy will be discussed.
The use of TNF-alpha antagonists has revolutionized therapy in patients with immune diseases, including rheumatoid arthritis, and inflammatory bowel disease. Among initial responders to TNF-alpha antagonists, response vanishes over time in approximately half of them. The most common cause of nonresponse is the production of antibodies against the TNF-alpha antagonist, which neutralize the effect of the drug. Most patients developing treatment failure are managed empirically by increasing the dose, or shortening dose intervals, without identifying the mechanism for loss of response, or investigating if the symptoms are due to causes other than inflammation. An alternative to this empiric strategy is to use laboratory testing to measure TNF-alpha antagonist drug and antibody levels, to select a new treatment based on the most likely mechanism responsible for loss of response. Testing for drug levels and antibodies to TNF-alpha antagonists is one of the fastest growing areas in clinical laboratory. Several methodologies are available for measuring levels of TNF-alpha antagonist drug and anti-drug antibodies. The objectives of this lecture are: 1) to describe the methods available for measurement of TNF-alpha antagonists and anti-drug antibodies levels; 2) to identify the clinical indications for which measurement of TNF-alpha antagonists and anti-drug antibodies levels are applicable; and 3) to provide guidance on interpretation of test results.
MB-003 is a cocktail of 3 monoclonal antibodies, originally developed for the treatment of Ebola Virus Disease. These mAbs bind to non-overlapping epitopes on Ebola virus glycoprotein, and one of the mAbs is also reactive with Sudan, Reston, and Tai Forest virus. The mAbs have been chimerized with a human constant region and expressed using a Rapid Antibody Manufacturing Platform in Nicotiana benthamiana. Individually the mAbs protect mice from lethal challenge prophylactically and two days post-exposure at doses less than or equal to 5mg/kg. When cocktailed, MB-003 protects Rhesus macaques from lethal IM challenge: 1000 pfu, with 100% protection demonstrated when treatment was initiated 1 hour post exposure, 67%-100% protection when initiated 1 or 2 days post exposure, and 43% protection when administered after detection of virus by both RT-PCR and a sustained fever; triggers met 100-120 hours post exposure. The majority of treated survivors display minimal morbidity and no side effects were observed from the treatment regimen in any animals. To improve on efficacy, various combinations of antibodies developed by Public Health Agency of Canada - ZMAb - were used to optimize protection in rodents and primates. The resulting combination of antibodies ZMappTM demonstrated improved efficacy over Zmab or MB-003. It is the result of an unusual partnership involving Mapp Biopharmaceutical, Inc. and Kentucky BioProcessing working with the U.S. government, and Defyrus, Inc. working with the Canadian government. Data to date indicate ZMappTM can provide therapeutic efficacy in non-human primates at least 5 days after infection
Narcolepsy is a chronic sleep disorder linked to the HLA-DQB1*0602 haplotype and dysregulation of the hypocretin ligand-hypocretin receptor pathway. Narcolepsy was associated with Pandemrix® vaccination (an adjuvanted, influenza pandemic vaccine) and also with infection by influenza virus during the 2009 A(H1N1) influenza pandemic. In contrast, very few cases were reported after Focetria® vaccination (a differently manufactured adjuvanted influenza pandemic vaccine). With these epidemiological associations as a starting point, we will review the approach taken leading to the identification of an influenza viral epitope with high similarity to the human hypocretin receptor 2 and the observation of antibodies against this receptor in patients with narcolepsy and a history of Pandemrix® vaccination.
The immune system uses diverse mechanisms of self-regulation to enable it to respond to dangerous pathogens whilst avoiding damage to “self”. Amongst these, regulatory T-cells are prime players. These cells can be harnessed therapeutically by giving them, numerical and functional advantages over T-cells that mediate damaging immunity. I will give examples using antibody-based co-receptor and co-stimulation blockade, and also by lymphocyte depletion with antibodies such as CAMPATH-1H, when depletion is followed by “Physician-Guided Reconstitution of the Immune System” (PARIS). Therapeutic antibodies can themselves be immunogenic, and lessons about mechanisms underlying immunogenicity, have been learned from strategies used to induce tolerance to the CAMPATH-1H antibody. These will be discussed briefly.
The immune response plays an important role in fighting cancer; however, the tumor environment is immunosuppressive and limits effective anti-tumor immunity. A new and promising strategy of tumor immunotherapy blocks pathways used by tumors to inhibit anti-tumor immunity. This inhibitory strategy is called checkpoint blockade. One key immunoinhibitory pathway that inhibits tumor specific immunity is the PD-1 co-inhibitory pathway. This pathway consists of the PD-1 receptor and its ligands PD-L1 (B7-H1) and PD-L2 (B7-DC). The PD-1 pathway plays critical roles in maintaining immune control, and is a key mediator of T cell dysfunction (“exhaustion”) in cancer and chronic infections. This pathway is a promising therapeutic target in cancer. The remarkable effects of PD-1 pathway blockade in cancer demonstrate the key role of this pathway in inhibiting anti-tumor immunity. However, there are multiple co-inhibitory pathways that that limit T cell function, and these have become targets for cancer therapy. This talk will discuss the multifaceted immunoregulatory roles of PD-1 and its ligands in controlling T cell activation, tolerance and exhaustion. The role of the PD-1 pathway in cancer as well as therapeutic strategies that combine PD-1 blockade with other therapies also will be discussed.
Tim Hickling is currently investigating the immunogenicity of biotherapeutics at Pfizer. He obtained his Biochemistry degree (1995) and Immunology Doctorate (1998) from the University of Oxford. He carried out post-doctoral training at Glaxo and the UK MRC’s National Institute for Medical Research before taking up a lectureship at the University of Nottingham, UK. Tim has worked on various aspects of innate and adaptive immunology in infectious and inflammatory diseases. Tim joined Pfizer in 2007 and has worked on early stage vaccines and from early discovery to late stage development of biotherapeutics.
The novel coagulation factor VIII molecule, rVIII-SingleChain, is composed of covalently bonded heavy and light chains to improve product characteristics, and thus FVIII therapy by enhancing molecular stability. The two-chain endogenous FVIII represents a labile configuration, although 95% of circulating FVIII is complexed with von Willebrand factor (VWF). This complex determines FVIII stability, its half-life and was shown to influence its presentation to the immune system. Yet, the unmet medical need for an improved FVIII therapy, that offers a reduced potential for immunogenicity combined with a more convenient dosing frequency is driven by a high incidence of inhibitory antibody development and an inherently short half-life of FVIII. Non-clinical studies were conducted to investigate whether enhanced integrity can translate into improved pharmacological characteristics of rVIII-SingleChain in non-clinical models. In comparison to marketed two-chain, full-length rFVIII the binding affinity of rVIII-SingleChain for VWF and its stability were investigated in vitro. In animals, the PK properties of rVIII-SingleChain, its procoagulant activity and immunogenicity potential to induce anti-FVIII antibodies was explored. Compared to full-length rFVIII, rVIII-SingleChain displayed a high affinity for VWF and a markedly enhanced molecular stability. Similarly, rVIII-SingleChain had more favorable PK properties, and thereby showed a prolonged thrombin generation potential and procoagulant activity. Furthermore, in FVIII knock-out mice rVIII-SingleChain induced a lower anti-FVIII antibody response. This potential for reduced immunogenicity is investigated employing different ex vivo human immune model systems. These non-clinical results warrant further exploration whether such improved molecular properties may translate into clinical benefit in hemophilia A patients
Innate immune collectins such as surfactant protein A (SP-A), SP-D, mannose-binding lectin and CL-11 are antibodies of the innate immune system. These collectins exert varying responses in circulation and mucosal surfaces, and certain collectins are known to improve immunogenicity of specific antigens. These collectins also interconnect microbes, antigens and dying cells to immune cells. However, the therapeutic potential of collectins has not also been fully explored. I will highlight the untapped therapeutic potentials of these proteins. Recent studies have established that innate immune cells such as neutrophils release neutrophil extracellular traps (NETs). Emerging evidence indicate that NETs are major regulators of immunogenicity in many infectious, inflammatory and autoimmune diseases. Regulating NETosis is an important aspect in treating these diseases. I will highlight the advances and therapeutic potentials of collectins and the prospective of regulating NETosis for altering immunogenicity and treating inflammatory diseases..
Interferon beta has been successfully used to treat multiple sclerosis since 1993, with a variety of administration routes and frequencies among the approved products. Interferon beta-1a IM is administered on a weekly schedule, and has an established safety and efficacy profile over 1.7 million patient years of exposure. Peginterferon beta-1a is a novel molecular entity with covalent attachment of a single PEG moiety to interferon beta-1a, and was developed with the intent to reduce dosing frequency while maintaining the safety and efficacy profile of interferon beta-1a IM. In Phase 1 trials in healthy human volunteers, peginterferon beta-1a demonstrated an extended half-life of 2-3 days compared to the 1 day observed with interferon beta-1a IM, resulting in greater exposure. Peginterferon beta-1a also generated a higher magnitude and a longer duration of pharmacological response; neopterin elevation induced by peginterferon beta-1a was more persistent compared with interferon beta-1a IM. The longer half-life and extended pharmacodynamic effect enabled dosing the therapeutic every 2 weeks and every 4 weeks in the pivotal phase 3 clinical study, as opposed to more frequent administrations performed for the non-pegylated IFN beta-1a products. Pegylation has been associated with reduced immunogenicity rates for some proteins. In the pivotal Phase 3 trial, immunogenicity against the interferon moiety of peginterferon beta-1a was lower than previously reported for other non-pegylated interferon beta-1a therapies; the incidence of persistent neutralizing antibodies was less than 1%. The incidence of persistent anti-PEG antibodies was 3%. No discernible impact of immunogenicity on clinical efficacy - primary and secondary endpoints - and safety was seen in this trial after 2 years of chronic treatment.
Re-establishing immune tolerance requires antigen specific CD4+CD25+Foxp3+Treg not nTreg which are non-antigen specific. We studied pathways for activation of nTreg antigen, and identified one activated by Th1 and another by Th2 cytokines. The initial activation by antigen respectively requires IL-2 or IL-4. Within days, nTreg express receptors for other lineage cytokines, respectively IFN-γ and IL-12 or IL-5. The IL-2 and antigen activated nTreg we call Ts1 cells, and they express CD25, Foxp3, IL-5, IFNGR, IL-12Rβ2, but no IL-2, t-bet, IFN-γ or IL-5Rα. Antigen with IL-12, in the absence of IL-2, activates Ts1 cells to Th1-like Treg that suppress in an antigen specific manner, and delay fully allogeneic rejection. Th1-like Treg express Foxp3, t-bet, IFN-γ, IL-12Rβ2, IFNGR and IL-5, but no IL-2. Similar Th1-like Treg can be induced by IFN-γ and IL-27. The IL-4 and antigen induced nTreg we call Ts2. Ts2 express CD4, Foxp3, IFN-γ, IL-5Rα, but no IL-2, IL-4, IL-5, Gata-3 IFNGR or IL-12Rβ2. Reculture of Ts2 cells with IL-5 and antigen, in the absence of IL-4, induces Th2-likeTreg expressing CD25,Foxp3,IL-5Rα,IL-4,IL-5,IRF4, but not T-bet,IL-2,IFN-γ, IFNGR. Ts1 and Ts2 cells can be generated in vitro in 3 days, are antigen specific and have greater than 10x suppression in vitro and in vivo than nTreg. Th1 and Th2 like Treg can be generated in vitro in 7 days and have 1000x greater capacity to suppress in vitro than nTreg. Therapy with IL-5 promotes Ts2 and Th2 like Treg and can suppress auto and alloimmune responses. These pathways can generate antigen specific Treg in vitro for use in therapy. Therapy with IL-5 enhances the Th2 pathway to promote antigen specific tolerance. We will discuss the potential therapeutic options for utilizing these antigen specific Treg activation pathways to restore
The Innovative Medicine Initiative’s Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk (ABIRISK) project has made significant progress in its second year. This presentation will provide an overview of current developments with a focus on anti-drug antibody and neutralizing antibody assays provided for the analysis of retrospective and prospective cohorts.
Immunogenicity risk management includes risk assessment and risk mitigation strategies. Knowledge about immune mechanisms is critical for improving the accuracy of risk assessments as uncertainty in knowledge about a risk factor increases its risk categorization. This talk will explore the impact of sub cutaneous, intra venous, and oral routes of exposure on the immunogenicity of therapeutic proteins.
The regulation of immune responses to self and foreign antigens is vitally important to limit immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune and allergic conditions can be reinforced by tolerance induction with peptide epitopes; as yet, however, the mechanism is not understood. Peptides must mimic naturally processed epitopes Repetitive administration of soluble peptide induces peripheral tolerance in models of autoimmune disease and allergy. This is characterised by the generation of anergic, IL-10 secreting CD4+ T-cells with regulatory function. CD4+ T-cells become anergic following their first encounter with peptide. The loss of proliferative capacity correlates with a cytokine switch from a pro-inflammatory to a phenotype characterised by secretion of the anti-inflammatory cytokine IL-10. IL-10 Treg cells suppress dendritic cell maturation, prevent Th1 cell differentiation and thereby create a negative feedback loop for Th1 driven immune pathology. These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen. Similar findings relating to Th2 mediated immune responses will be discussed.
Interferon beta (IFNb) has been used in the treatment of MS for 20 years. There are three different recombinant preparations available. IFNb-1a is identical to the human form (produced in Chinese hamster ovaries) and IFNb-1b is different to humans lacking an amino acid (AA), containing one AA substitution, and lacking glycosilation. Of the two IFNb-1a preparations one is applied subcutaneously and one intramuscularly. On top of that, the formulations are different and the degree of aggregation varies, with the i.m. IFNb-1a showing almost no aggregation in contrast to the other product containing relatively high proportions of drug aggregates. Not surprisingly the frequency of anti-drug antibodies (ADA) differs substantially between these preparations with a range of 50% to almost 100% on the total ADA level and 5% to 40% on a neutralizing antibody (NAB) level. One of the questions arising is whether neutralization of IFNb simply depends on the total ADA titers or if it depends also on other factors. In fact it turned out that interferon antibodies in NAB positive patients undergo affinity maturation, show differences in epitope recognition as compared to patients without NAB, and IgG2 and 4 subtypes predominate in NAB positive samples. Overall, there is a good correlation between total ADA and NAB titers, but there is a weak correlation in low to intermediate NAB titer ranges, and a strong correlation in high NAB titer patients. Taken together, various factors contribute to neutralization of IFNb which cannot be explained merely by the amount of total ADA. This is supported by the observation that some patients with low ADA titers display a high degree of IFNb neutralization and vice versa
XTEN is a family of unstructured polypeptide with biophysical properties similar to PEG, but is superior to PEG in that it is highly versatile and biodegradable. In this talk, we will address utilizing XTEN in engineering the next generation blood factors with superior PK/PD and immunogenicity profile
It has recently become clear that different B cell subsets contribute to both the pathogenesis of immune disorders, as well as the maintenance of tolerance, and are therefore implicated in the development of autoimmunity. Patients with rheumatoid arthritis (RA) and systemic lupus erythematosus are characterized by an increase in pathogenic B cells (e.g. memory), and a decrease or functional impairment of regulatory B cells (Bregs), which promote tolerance via the production of interleukin-10. In humans, we and others have shown that B cells with suppressive capacity are enriched in the CD19+CD24hiCD38hi compartment. These cells can interrupt T cell-B cell positive feedback, suppress effector T cell proliferation, and induce regulatory T cell differentiation. Yet conclusive markers for the identification of a pure Breg population, distinct from those with pathogenic capacity, have not been found. Here we describe a new immunophenotyping platform, with which we can screen the expression of 242 cell-surface markers in parallel by flow cytometry. This has enabled us to generate a surface marker “signature” for different B cell subsets, and provided us with a phenotypic counterpart to gene array data. A major goal in the development of biopharmaceutical drugs is to predict which patients are likely to respond with anti-drug antibodies (ADAs) – one of the principal drawbacks of biological therapies. We will implement our method to compare B cell subset signatures in a longitudinal cohort of RA patients treated with anti-TNFa in order to identify predictive markers for those patients who will become ADA positive.
Competition for limited, cell extrinsic survival factors is a general feature of the peripheral selection checkpoints involved in B lymphocyte maturation, activation and memory. Perhaps the best-characterized example involves BLyS family cytokines and receptors, which governs survival and differentiation within B cell subsets. Discovery of the BLyS cytokine and receptor family has proven a watershed event, significantly advancing our understanding of B lymphocyte selection and homeostasis. This family includes two ligands, BLyS (also termed BAFF) and APRIL; as well as three receptors, BR3 (also termed BAFFr), TACI, and BCMA. Members of this molecular family play critical roles in maintaining immunological tolerance, by impacting selection and survival in nearly all B cell subsets. Cells in the transitional and mature, pre-immune B lineage subsets rely on BLyS signals via the BR3 receptor for survival. In contrast to tolerogenic elimination at the BM immature B cell stage, the transitional checkpoint displays plasticity by integrating BCR-mediated selection with BLyS-mediated peripheral B cell homeostasis. Thus, when BLyS levels are elevated, transitional selection is “relaxed,” so that B cells that would normally be negatively selected instead survive to join mature naïve pools. Mounting, evidence suggests analogous competitive checkpoints for both germinal center B cells and plasma cells. In contrast to pre-immune pools, B cells in recently activated and antigen-experienced subsets shift their BLyS receptor profiles and hence their ligand reliance. For example, short-lived antibody-forming cells adopt a TACI dominated BLyS receptor signature, whereas germinal center (GC) B cells profoundly down-regulate TACI but retain BR3. The lack of TACI on GC B cells leads to a paucity of retained BLyS in the GC, such that the sole local source of BLyS in the GC is the TFH cell. Moreover, BLyS expression by GC TFH is crucial for appropriate GC evolution, since efficient affinity maturation fails if T cells are BLyS deficient. Finally, long-lived plasma cell pools express BCMA, shifting reliance to APRIL. Considered together, these observations suggest that deliberately altering BLyS levels might be a means for manipulating B cell repertoire selection in order to (1) restore self-tolerance in autoimmunity, (2) remodel the repertoire to accommodate neo-self antigens introduced through biologicals, transplantation and gene therapy, or (3) temporarily expand B cell repertoire diversity to reveal novel, therapeutically useful specificities.
Although most Anti-Drug Antibodies (ADA’s) are innocuous, some low abundance or low frequency ADA’s may pose a significant clinical risk which is difficult to evaluate at the serum level. Trellis’ established technology platform for efficient cloning of rare B-cells enables deconvolution of serum into its component monoclonal ADA’s. Our computational B cell epitope prediction technology accelerates cloning ADA’s against all epitopes on the protein when a high quality structure is available. The combined computational and empirical technology provides a rational route to immunological silencing of potentially problematic epitopes.
The presence of host cell and drug impurities in biologic drug preparations can have repercussions on drug safety and efficacy by triggering innate immune responses that may amplify adaptive immune responses to a protein therapeutic. Effects of innate immune response modulating impurities (IIRMIs) depend on the a variety of parameters, such as the quantity and type of the impurity as well as the dose and route of drug administration. Challenges in developing methods to quantify and characterize the qualitative nature of an innate immune response to biologic drug preparations will be discussed, as will the implementation of such methods in assessing the potential impact of manufacturing decisio
As next generation biologics such as novel scaffolds, antibody fragments and fusions progress towards the clinic the need to understand and predict potential immunogenicity issues is a crucial consideration. De-risking therapeutics at an earlier stage in development ultimately decreases more costly pipeline attrition at later stages of drug development. The Shark VNAR Development team (Elasmogen) have optimised both immunized and synthetic platforms to isolate novel therapeutic VNAR domains (the smallest binding site so far identified in nature). Elasmogen are currently undertaking an immunogenicity assessment of our lead half-life extension bio-tool, E06, with a view to increasing our understanding and to mitigate any downstream risks associated with adverse immune responses in any “first-in-man” studies. E06 is derived from an immunised shark and its crystal structure in the presence of antigen has been successfully determined. It binds with picomolar affinity to albumin and importantly extends the serum half-life of fused N-terminal and C-terminal binding domains from hours to days across rodent and non-human primate models of PK. Its predicted half-life extension in man is 19-20 days. In vivo studies to date, including a multi-dosed study in cynomolgous monkeys, have provided no evidence of immunogenicity. This aside, we felt it important to undertake a program of humanisation to minimise any downstream regulatory issues when utilising native non-human domains. This talk will cover our E06 humanisation strategy, in silico and in vivo immunogenicity data and work completed with ProImmune to date, to aid final humanised candidate selection for later stage pre-clinical development and eventual clinical studies.
Dr. Federico Mingozzi details the AAV-2 gene therapy model in Phase I/II trial hemophelia patients. He showed that a loss of human FIX expression was associated with expansion of capsid-specific CD8 T cells and demonstrated a dose-dependent T cell activity against the vector.
The immunogenicity profile of a biotherapeutic is determined by a multiplicity of factors ranging from product related, patient (host) related, bioanalytical to process or manufacturing related factors. This creates a complex situation that does not allow direct correlation of such risk factors to the observed incidence of immunogenicity. Therefore, a mechanistic understanding of how these risk factors individually or in concert influence the overall incidence and risk of immunogenicity is crucial to design the best benefit/risk profile for a given biotherapeutic in a given indication. In light of the observations that this field of Predictive Immunogenicity has not progressed sufficiently in the past few years, several forums have focused on investigating the impediments and the reasons behind these impediments that contributed to the lack of progress. The Predictive immunogenicity survey and the Open Forum that was conducted under the AAPS NBC banner provided insights into some of the gaps that exist in this regard. One of the key observations was that almost all biotech pharma scientists were familiar with the predictive tools but very few of them were actually using it. This in fact results in very little data coming in the public domain. That cascades into an impression of poor reliability of these tools in predicting clinical relevance of immunogenicity, culminating into very few companies using these tools. This chain must be broken if this field has to progress. At present prediction of immunogenicity is not requested by the regulatory agencies. The lack of such information does not preclude a sponsor from filing and getting approvals of biotherapeutics. Most sponsors are effectively managing the associated immunogenicity risks in clinic for their drugs, with a variety of strategies from medications to monitoring. So then why would a sponsor spend time and money to gather such data? We believe this disconnect is primarily due to the lack of an established “Value Added Proposition” for the employment of predictive immunogenicity exercises in drug development. Investments will flow in this area if the science, the collaborations and the data clearly demonstrate how these predictive immunogenicity studies can directly influence the commercial success or failure of a drug. Therefore we have made an attempt to illustrate through examples the value added proposition by collecting information from literature and various sources in the industry to highlight how predictive immunogenicity efforts can impact the bottom line in due course of time. It is this sort of information that needs to be disseminated to Sr. Management in the Pharma and the Biotech industry who can provide for the funding and influence the course of this discipline leading to more case studies and consolidation of the knowledgebase to further the science of making safe and effective drugs
The biotherapeutics field continues to expand and introduce both new innovative therapies, as well as biosimilar and improved versions of marketed biotherapeutics into the clinic. In this environment, risk of inducing unwanted anti-therapeutic immune responses (immunogenicity) remains a concern due to the potential to affect safe or efficacious use, or affect development costs, regulatory pathways, or competitive position in the market. Because of the potential risks to product safety and survivability, there is increasing pressure to develop improved methods to assess risk of unwanted immunogenicity and its consequences in order to inform risk mitigation strategies and business decisions. Well known factors contributing to risk for development of immunogenicity include product-, patient or disease-, and treatment regimen-related attributes. Most likely, multiple factors contribute simultaneously to overall risk. This presentation will overview the sources of risk and how incorporation of new risk assessment tools could provide more accurate assessment to improve risk mitigation
Genetic analysis of point mutations is a valuable tool in elucidating protein structure/function relationships. We have designed and implemented a new analytical platform based on efficient gene synthesis and high throughput DNA sequencing to generate and analyze in parallel the effects of many hundreds of point mutations on protein binding. Our MapEng™ protein analysis platform guides the design of biobetter molecules with a variety of improvements, including enhanced affinity, reduced immunogenicity, modified cross reactivity and pH sensitivity. We have used this system to assess the effects on affinity of all possible point mutations in and around heavy chain CDR3 of Avastin. Up, down and neutral mutations with respect to binding were discovered or confirmed published results. These results demonstrate the utility of MapEng™ for creating biobetter therapeutics
Immune responses to biotherapeutics are increasingly recognized as a risk to the success of projects and products in the Biopharmaceutical industry. These risks have various consequences ranging from project delays in the preclinical phase, to increased regulatory burden following clinical anti-drug antibody (ADA) observations, and in extreme cases to project termination. Many factors contribute to the overall risk of inducing ADA, for example route of administration, protein aggregates, T cell responses, drug pharmacology, impurities and patient status. Regulatory authorities offer guidance as to the assessments of immunogenicity risk they expect sponsors to undertake prior to commencement of clinical trials. In addition, appreciating the risks associated with projects can add confidence for an organization to commit funds to a clinical trial. Applying recently developed and improved tools can generate more confidence and hopefully allow for a better chance of success. The knowledge gained across a range of Biotherapeutic projects will allow general and product specific risks to be recognized and addressed. At the frontier of this effort we are investigating a systems biology and mechanistic modeling approach to predicting immune responses. Broader understanding of the immune system offers hope for safer and more efficacious products. I will give an overview of the issues and the tools to investigate immune responses for immunogenicity. Dr Tim Hickling is currently investigating the immunogenicity of Biotherapeutics at Pfizer. He obtained his Biochemistry degree (1995) and D. Phil (1998) from the University of Oxford. He carried out post-doctoral training at Glaxo and the MRC’s National Institute for Medical Research before taking up a lectureship at the University of Nottingham. Tim has worked on various aspects of innate and adaptive immunology in infectious and inflammatory diseases. Tim joined Pfizer in 2007 and has worked on early stage vaccines and from early discovery to late stage development of biotherapeutics.
A major concern in the development of biological therapeutics is the induction of antibodies against the biological in patients undergoing therapy. Such antibodies can adversely affect the safety and efficacy of a therapeutic product. A rigorous assessment of immunogenicity, based on state of the art measurement and characterization of antibodies, to understand the immunogenicity of the product is therefore required. A well considered strategy for immunogenicity assessment which includes a risk-level based bioanalytical testing strategy and a management plan for related adverse events is essential. The CHMP of the EU has produced a general guideline on immunogenicity assessment of biotechnology-derived therapeutic proteins and also a draft guideline on immunogenicity assessment of monoclonal antibodies intended for in vivo clinical use. Other CHMP guidelines also contain advice on specific aspects of unwanted immunogenicity. These will be discussed during the presentation. Robin Thorpe PhD, FRCPath, is Head of the Biotherapeutics Group at the National Institute for Biological Standards and Control (NIBSC). Recent interests include the unwanted immunogenicity of biologicals, development of improved bioassays for cytokines, the immunology of monoclonal antibodies and cytokine contamination of biological products. He attends meetings of the Biologicals Working Party & Biosimilars Working Party of the CHMP at the EMEA. He is a member of the British Pharmacopoeia Commission (MHRA) Expert Advisory Group NOM, the British Pharmacopoeia Panel of Experts on Biological and Biotechnological Products and chairman of the Working Group on Monoclonal Antibodies of the European Pharmacopoeia. Dr. Thorpe is a member of the Biologicals & Vaccines Expert Advisory Group of the CHM. He is the Chairman of the IUIS nomenclature subcommittee for chemokines and the standardisation and nomenclature committee of the International Cytokine Society. He is a biologicals advisor to the WHO INN programme. Dr. Thorpe is an author on over 200 publications in scientific journals and books. He is an associate editor for the journal Cytokine, Biotherapeutics section editor for Biologicals and editorial board member of the Journal of Immunological Methods & Current Analytical Chemistry.
It is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high affinity antibodies against proteins. CD4+ T cells recognize peptides (epitopes) presented by MHC-class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC-class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC-class II-peptide complex. We used a new humanized hemophilic mouse model to study the regulation of immune responses against factor VIIII (FVIII) by immunodominant FVIII peptides presented by HLA-DRB1*15:01. This new mouse model carries a knockout of all murine MHC-class II molecules and expresses a chimeric murine-human MHC-class II complex that contains the peptide binding sites of the human HLA-DRB1*15:01. Using T-cell hybridoma technology, we identified 8 FVIII peptide regions that contained CD4+ T-cell epitopes presented by HLA-DRB1*15:01 to CD4+ T cells during immune responses against FVIII. Most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays. Despite their promiscuous binding behaviour, the kinetics of peptide binding to the HLA-DR proteins investigated differed substantially. Katharina Steinitz, PhD, is a scientist in the Department of Immunology within the division of Hemophilia and Hematology at Baxter Innovations GmbH. She received her first degree in Molecular Biology from the University of Vienna, Austria, and studied for her PhD in Immunology at the Technical University of Vienna. Her current research focuses on the immunogenicity of therapeutic proteins and the search for new approaches to prevent these unwanted immune responses.
Biotherapeutics have been shown to be effective therapies which demonstrate significant target specificity and may confer multiple effector functions: for example receptor-blocking or -antagonism combined with target cell cytotoxicity of a monoclonal antibody. However, the use of biotherapeutics in the clinical setting has been complicated by the development of immunogenicity (anti-drug antibodies) against the therapy. While it is important to remember that the presence of ADA in patients often has no detrimental effect, there may be an impact upon PK, efficacy or even safety. Thus numerous pre-clinical models have been developed with the aim to better understand mechanisms involved in immunogenicity induction and to use this information to reduce the risk of immunogenicity. Technologies, including MHC-associated peptide proteomics and novel in vivo models will be compared and contrasted in this talk, with their potential uses and limitations discussed. Heather Hinton is the head of ImmunoSafety at F. Hoffmann La Roche where, in addition to assessing and mitigating immunosafety liabilities of therapeutics, she has an interest in the immunogenicity of biotherapuetics. Heather obtained her PhD in Pharmacy and Pharmacology from the University of Bath, followed by a PostDoc at Cancer Research UK and the University of Dundee working in adaptive immunity. Following this she moved to a small biotech company specializing in therapeutic vaccines development, where her involvement in safety of therapeutics was initiated.
There are no licensed vaccines or antivirals for the treatment of Venezuelan Equine encephalitis virus (VEEV) infections. A medical countermeasure is required for accidental exposure or deliberate release (for example, bioterrorism or biowarfare). Neutralising monoclonal antibodies have previously been shown to prevent or mitigate disease in small animal models. Furthermore, protection has been achieved against aerosol challenge with multiple strains of the virus. We have humanised one of the more broadly reactive murine monoclonal antibodies by the process of Complementarity Determining Region (CDR) grafting using human germline IgG frameworks. Studies with this candidate medical countermeasure have fallen into three areas. Firstly, a theoretical assessment of the impact of availability of an antibody therapy against environmental release of VEEV was performed utilising the HPAC modelling system. Secondly, we have examined the effect of humanisation on the potential to cause adverse events in humans using ex vivo inflammatory cytokine and T cell proliferation assays. Thirdly, the retained biological properties of the humanised antibody have been determined by assessing in vitro binding to different VEEV strains and neutralisation, and ability to prevent death in the mouse model of disease. We are able to demonstrate that the humanised antibody is able to provide protection against lethal subcutaneous and aerosol challenge with the virulent Trinidad Donkey strain of VEEV, and describe the therapeutic window in which it is effective. Crown copyright, Dstl 2011. Dr David Ulaeto obtained his B.Sc. (Biological Sciences, 1984) and Ph.D. (Cancer Research, 1988) from the University of Birmingham, UK. He did post-doctoral training in the Department of Pathology, at Washington University School of Medicine in St. Louis from 1988-1991, and was an Assistant Professor of Microbiology at Oregon State University from 1992-1995. In 1995 he moved to Dstl Porton Down, where he continues as a Principal Scientist. His research interests are in virology and since 2000 he has been an advisor to the WHO on variola virus research. Since 2009 he has advised the FAO and OIE on sequestration of rinderpest virus.
Protein therapeutics may elicit unwanted immune responses in some subjects, and this can compromise product safety. This talk will discuss some of the causes and consequences of unwanted immune responses to biologics. The talk will also review what tools, technologies and data sets may be deployed to assess immunogenicity and to enable informed decision making during biotherapeutic product and process development. Valerie Quarmby is a Staff Scientist in the Department of BioAnalytical Sciences at Genentech. At Genentech, Dr. Quarmby has developed bioanalytical methods & strategies to enable IND, BLA and related filings for: Nutropin, Xolair, Raptiva, Rituxan, Avastin, Lucentis, Perjeta, Kadcyla and Tecentriq. She has also developed bioanalytical methods, platforms and strategies for many of the therapeutic proteins in the gRED drug development pipeline. Valerie also consults on bioanalytical and related strategies across the Roche group. She is the past Chair of the AAPS Therapeutic Protein Immunogenicity Focus Group and a member of the 2010-2015 USP Immunogenicity Testing Expert Panel. Valerie is the co-author of several AAPS sponsored industry guidance documents and many publications in peer-reviewed journals. Valerie Quarmby holds a Ph.D. in Hormone Physiology from the Imperial Cancer Research Fund and the University of London in England. Dr. Quarmby was a Postdoctoral Visiting Fellow at the US National Institutes of Health, and then joined the Laboratories for Reproductive Biology and the Department of Pediatric Endocrinology at UNC-Chapel Hill. Prior to joining Genentech, Dr. Quarmby worked in the field of clinical diagnostics at Bio-Rad Laboratories and at Endocrine Sciences/Esoterix. In 2014, in recognition of her many contributions to the pharmaceutical industry, Dr. Quarmby was awarded AAPS Fellow Status.
Managing immunogenicity risk should be a core focus in drug development. ProImmune's CEO Nik Schwabe explains why.
