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Immunotechnology

Faculty of Engineering, LTH

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Doctoral thesis abstracts

Anne Ljungars: Phenotypic antibody discovery and mining of complex antibody libraries

(2020)

Therapeutic antibodies are the fastest growing class of drugs but suffer from a crowded target-space where many antibodies, against a few targets, are developed in parallel. The aim of this thesis is to find methods to enable discovery of novel antibody-target combinations and thereby increase the target-space. A strategy to achieve this is to use phage display antibody libraries and selection on whole cells in a phenotypic antibody discovery process, where antibodies with the desired function are isolated without knowing the target. Thereafter the targets for the functionally interesting antibodies are determined. In theory, this strategy allows discovery of antibodies against all receptors on the cell surface (potential targets). However, typically only the most abundant antibodies binding a few, commonly highly expressed, targets are identified. Methods to mine the generated antibody pools to allow discovery also of rare antibodies are therefore needed. In this thesis, I establish a phenotypic antibody discovery platform and apply it on primary cancer cells from patients with chronic lymphocytic leukemia, CLL. Novel antibody-target combinations, with enhanced cytotoxicity, both in vitro and in vivo, compared to the standard of care were discovered. Furthermore, I showed that by applying various deep mining methods on the generated antibody pool, additional antibodies could be discovered. These rare antibodies bound new epitopes on the target cells, either on previously discovered, or on entirely new targets. In addition, I demonstrate through analysis by next generation sequencing, NGS, that the number of receptors on the cell surface has a major impact on antibody enrichment in the selection process. By following clonal enrichment with NGS, both abundant and rare antibodies were discovered. This allows the conventional immunochemical screening of a phage display antibody discovery process to be by-passed.

The discovered antibodies can be used in many different applications. For some of these applications, bispecific antibodies, designed to bind two different antigens, can have improved efficacy through e.g. increased specificity compared to conventional monospecific antibodies. However, many of the current bispecific formats, especially those similar to a conventional IgG, are difficult to construct. In this thesis, I create a new type of bispecific antibody, the Tetra-VH IgG. This antibody format contains four variable heavy (VH) domains, is tetravalent, and binds two antigens simultaneously on each Fab arm. The Tetra-VH IgGs potentially show enhanced binding and functional properties compared to conventional bivalent IgGs.

In summary, the technologies I describe in this thesis will be important in future antibody discovery efforts as they enable a broad repertoire of antibodies against novel targets, of both high and low receptor density, to be discovered and used for treatment of various diseases.

For additional information, please contact Mats Ohlin.

Tim Lindberg: Mechanistic Toxicology - Molecular interactions and underlying mechanisms of dendritic cell activation in skin sensitization

(2019)

Allergic skin diseases caused by low molecular weight chemical compounds are a major health concern among the general population, especially in certain occupational groups. To limit exposure to such compounds, it is important that thorough risk assessments are performed before products reach the consumer market. Traditionally, such risk assessments have been based on animal tests, but due to several factors, including legislations and ethical concerns, the use of animal testing has become increasingly unjustifiable. In this context, one focus in predictive toxicology has been the development of alternative methods to replace animal experiments. Although several alternative methods have been validated for regulatory decision making, they lack certain endpoints for complete risk assessments, and molecular mechanisms underlying skin sensitization are still not entirely understood. The work described in this thesis aims at increasing our knowledge about the mechanisms in dendritic cell activation in response to skin sensitizers and to further develop a dendritic cell-based in vitro assay.

In Paper I, we investigated the microRNA regulation in response to stimulation of a DC model with structurally similar rubber sensitizers. The changes triggered by the rubber sensitizers in both mRNA and microRNA expression suggest a chemical-specific regulation, despite the structural similarity of the rubber sensitizers. In Paper II, the skin sensitizing properties of herbicidal formulations are investigated. Here, we predicted the herbicide glyphosate as a non-sensitizer, while the co-formulant polyethylated tallow amine and two glyphosate-based formulations were predicted as sensitizers. Additionally, we also investigated the proteomic alterations in response to these chemicals and formulations, and identified cellular responses associated with the differentially expressed proteins. In the last two papers, Paper III and Paper IV, we present additional applications for the in vitro assay setup used in this thesis. In Paper III, we identified a biomarker signature for the prediction of skin sensitizer potency, demonstrating a balanced accuracy of 78% targeting three potency classes, i.e., 1A (strong sensitizers), 1B (weak sensitizers) and no cat (non-sensitizers). In Paper IV, we applied a statistical method, the conformal prediction framework, to investigate the predictive boundaries of the in vitro assay, and concluded, based on 70 chemicals, that the assay can be applied to a large chemical space.

In conclusion, the work presented here can contribute to a better understanding of the mechanisms underlying the immunological response to skin sensitizing chemicals, which together with state-of-the-art predictive assays could be used for improved risk assessment of chemical compounds and to develop tools for prevention and treatment of allergic skin diseases.

For more information please contact Malin Lindstedt.

Valentina Siino: Phosphoproteomics to tackle brain disorders: Insight into the murine brain phosphoproteome

(2018)

Brain disorders affect the normal functionality of the brain causing inability in controlling certain behaviours. In the last two decades, despite the technological advancement, many biological questions concerning brain mechanisms and neural networks remain still unclear. Thus, the integration of multiple technologies could help on clarify several aspects of neurobiology.

The aim of this thesis was to study the function of certain proteins and protein-protein interactions in murine brains by using mass spectrometry-based proteomics. Since one of the main switching mechanisms in cells is the phosphorylation of proteins, the phosphoproteome of murine brains was specifically studied.

The results presented in this thesis let to the identification of phosphoproteins implicated in molecular pathways that could be responsible for brain impairment during certain conditions like obesity (Paper I and III), anxiety and depression (Paper III) and Parkinson´s disease (Paper IV). The findings of this thesis remarkably contribute to the proteomic and neurobiology fields and set phosphoproteomics as a powerful tool for the study of neuroscience.
    

Mattias Brofelth: Technological advancements in affinity proteomics : From planar antibody microarrays towards a solution-based platform

(2018)

Proteomics has the potential to deliver disease-associated biomarkers that could provide diagnostic, prognostic and predictive information to enable precision medicine. Affinity proteomics, most commonly based on antibodies and their ability to specifically capture target proteins, has emerged has a valuable tool in biomarker discovery. Our group has developed a recombinant antibody microarray platform that can be used for protein expression profiling of serum samples to define multiplex biomarker signatures. This thesis is focused on antibody engineering and assay development to further improve the current microarray platform and also present proof-of-concept for a novel solution-based platform.

In Paper I we evaluated the novel detection reagent PID in search for increased signal-to-noise ratio and improved sensitivity of the microarray assay. PID is a fluorophore-packed nanoparticle and was here used to replace the currently employed single fluorophore molecule. The result showed that it was possible to use PID as a detection reagent in our assay and even higher signals were achieved, although accompanied by a heterogeneous background that will require further optimization.

In Paper II and III we explored the Dock’n’Flash method for site-specific conjugation to enable oriented immobilization or functionalization of scFvs. Immobilizing the scFvs in an oriented configuration on the slide could lead to increased sensitivity and performance of the microarray assay. Functionalization could enable novel scFv applications. The scFvs were equipped with the unnatural amino acid pBpa and photocrosslinked to beta-cyclodextrin on a coated slide or in solution. Proof-of-concept was demonstrated for one scFv in Paper II and the study was expanded and the pBpa position optimized in Paper III.

In Paper IV we sought to overcome some inherent limitations associated with planar microarrays for global serum profiling by developing the solution-based MIAS platform. In MIAS, proteins were displayed on beads and quantified via DNA-barcoded scFvs using next generation sequencing (NGS). Sortase A was used to site-specifically conjugate the oligonucleotide barcode to scFvs. Proof-of-concept for the assay steps was demonstrated using barcoded scFvs targeting three different serum proteins.

In summary, the work presented in this thesis can be used to improve the performance of our current antibody microarray platform and also provides the first steps towards a novel solution-based platform. This could in turn enable improved development of disease-associated protein biomarkers.
    

Andy Forreryd: GARD-Genomic allergen rapid detection. From biomarker discovery towards a state-of-the-art testing platform for chemical sensitizers

(2017)

Chemical hypersensitivity reactions induced in the skin or in the respiratory tract are important health concerns and develops following repeated exposure to certain chemicals, termed sensitizers. To prevent such hazardous compounds from entering the consumer market, legal frameworks within EU require chemicals to be tested for their capacity to induce hypersensitivity. This type of testing has traditionally been performed in animals, but a recent paradigm shift has been initiated to promote the development of in vitro alternatives. However, currently, such proposed strategies can only be used for identification of skin sensitizing hazard, and are unable to inform on other endpoints of regulatory concern, such as skin sensitizing potency and respiratory sensitization.

The GARD assay was developed as a multiparametric alternative to current in vitro tests. The assay measures chemically-induced changes within a predictive biomarker signature of 200 genes using transcriptome-wide microarray profiling and has proven to be highly accurate for identification of skin sensitizers. The overall ambition of the work presented in this thesis has been to utilize novel transcriptomic technologies, advanced computational tools, and powerful machine learning strategies to further transform GARD into a versatile and highly standardized tool for regulatory decision-making.

The first part of this thesis addressed the need for a more versatile test platform capable of targeting additional toxicological endpoints. By measuring complete transcriptomes of cells following chemical stimulations, two separate biomarker signatures could be identified and demonstrated to have potent ability to predict respiratory sensitizing properties and skin sensitizing potency, respectively. The second part of this thesis addressed modifications to current GARD protocols to facilitate progression from biomarker discovery into a highly standardized tool for chemical risk assessment. A novel streamlined workflow was presented, where gene expression measurements had been transferred from transcriptome-wide profiling into the NanoString nCounter platform, measuring only genes in the biomarker signatures, which enabled for increased sample throughput, simplified protocols, and reduced assay costs in a format adapted to routine testing.

To conclude, by combining the novel biomarker signatures identified in this thesis with the previous biomarker signature for identification of skin sensitizers, the GARD platform demonstrates a unique possibility to simultaneously screen for skin sensitizing hazard and potency, as well as respiratory sensitizing properties in a single test sample. Following the introduction of a novel pipeline to progress from initial biomarker discovery into a highly standardized testing format, the results presented in this thesis shows that GARD is a state-of-the-art platform ready to replace animal experimentation for testing of chemical sensitizers.

For more information, please contact Malin Lindstedt.
  

Emila Kurbasic: Landscaping the cell surface proteome of breast cancer: Following pathways through organelles to the plasma membrane

(2017)

Breast cancer is one of the most common cancers in women. It is most commonly treated by the surgical removal of the tumour in combination with (neo)-adjuvant therapy (hormone, chemo- or radio- therapy before or after surgery). However, a large number of patients are over treated, with approximately 60% being given adjuvant therapy when already cured by surgery alone. This causes many undesirable side effects and cost hence, there is great need for new diagnostic and prognostic methods to decide if patients are in need of adjuvant therapy.

A number of prognostic and treatment predictive factors have been established such as tumour size, hormone receptor status, histological grade and age. Hereditary predisposition to developing cancer can be a factor, with several high penetrance genes identified such as BRCA1 and BRCA2 genes as well as many as a dozen lower risk genes that are however additive in effect. Molecular subtyping has significant prognostic value allowing the differentiation of several subtypes having unique survival outcomes, particularly for tumours highly responsive or nonresponsive to hormonal or targeted drug therapies. Currently the prognostic and treatment predictive factors are mainly based on the primary tumour status, even though the distant metastases are the main reason for breast cancer related deaths. Thus, there is need for novel approaches with higher specificity and sensitivity in newly developed targeted therapies.

The aim of this thesis was to understand the changes in breast cancer tumour cells and tissues, by comparing protein expression levels in different conditions, using mass spectrometry. Attention was specifically on the analysis of patient samples, with pairs of primary tumours and metastases, in order to try to understand what happens to allow tumour cells to be able to metastasise and to identify novel molecular markers. Hence we also investigated the biological functions of proteins by integrating information about the processes and pathways in which these proteins take part in order to be able to better understand the contribution of different proteins to breast cancer development. Further we have explored molecular classification markers for breast cancer tumours into major intrinsic subtypes. Our results demonstrated a great overlap of subtypes, using gene expression and protein expression profiling. In conclusion, all our findings together show the great need for improved and early cancer detection and treatment as well as need for development and promises of personalized medicine.

For more information, please contact Peter James.
  

Petter Skoog: Molecular portraits of cancer: Discovery of biomarker signatures using affinity proteomics

(2017)

The use of antibodies as capture molecules in assays is a common practice. By either printing monoclonal single chain fragment variables (scFvs) on a solid support – recombinant antibody microarray – or attaching them to magnetic beads – Global Proteome Survey (GPS) – we can specifically capture and measure target proteins of interest. Using the recombinant antibody microarray, we target predominantly intact immunoregualtory proteins, and measure their expression patterns in clinical samples, such as cancer samples. With GPS, we target peptides derived from digested (cancer) samples to identify and quantify a variety of proteins. While antibody microarrays use one antibody per targeted protein, GPS targets peptides, where one antibody can bind hundreds of different proteins – i.e. one antibody per several target proteins. The latter is accomplished by designing antibodies against a short 4 to6 amino acid long peptide motif shared among these proteins. In this thesis, both of these methods have been used to decipher cancer biomarker signatures.

In paper I, the antibody microarray was used to examine the immunosignature of pancreatic ductal adenocarcinoma (PDAC), and how the profile of the immune response differed between cancer patients compared to both healthy and benign controls. We successfully identified an immunosignature of four to ten scFv antibodies that could classify PDAC from controls with high specificity and sensitivity.

In paper II we investigated with the antibody microarray how the immune response evolved with, and shaped, emerging cancer cells in patients later diagnosed with breast cancer using antibody microarray. By determining protein expression profiles for cases and controls, we identified several deregulated immunological proteins that reflected evolving breast cancer, up to two years before diagnosis.

In papers III and IV, we used both the antibody microarrays and GPS to decipher biomarker signatures to differentiate between histological grades in breast cancer tumors. With molecular signatures capable of classifying grade, we could also visualize the heterogeneity of intermediate grade 2 tumors. Grade 2 tumors have little clinical prognostic value, and a majority of samples are classified as grade 2. Results from paper IV showed that it might be possible to reclassify many of these samples as either grade 1 or 3, giving clinicians further information for optimal treatment selection.

In conclusion, in this thesis I have demonstrated the use of profiling parts of the immune response as a tool for surveying and classifying disease, by using antibodies as specific binders to capture and measure low and high abundant proteins.
  

Kristoffer Sjöholm: A holistic approach to host-pathogen interactions: Detecting the large to unravel the small

(2017)

Sepsis is one of the leading causes of mortality and morbidity in the world, and is an overreaction by the immune system due to pathogen invasion of the bloodstream. The interactions, particularly the protein interactions, between the host and pathogen are fundamental for the outcome of the disease. However, some of the protein interactions are unknown and those that are known have been studied as single entities. This thesis focuses on expanding the knowledge of these interactions. In addition, the thesis emphasises the analysis of every interaction at the same time. The identification and quantification of the whole network of interactions is central to determine the impact of single proteins, since the interactions can be co-dependent. The proteins of the human immune system are of particular interest since the pathogens have to avoid the immune system in order to survive in the host. Using state of the art techniques, the human plasma interaction proteome for several pathogens were determined and several novel interactions were discovered. For the gram-positive bacterium Streptococcus pyogenes, the interaction proteome was determined with higher accuracy than ever before. In conclusion, when focusing on the whole interaction pattern the specific details can be explained. The wider understanding of the field of host-pathogen interactions that is established with this thesis aids in future development of diagnostics and therapeutics in pathogen related diseases.
  

Leena Liljdahl: Exploring the diabetic kidney proteome. A junction of tissue and serum.

(2017)

Diabetic kidney disease is one of the major complications associated with diabetes. Approximately 25-40% of both type 1 and type 2 diabetes patients will acquire a kidney complication and despite global efforts to reduce the prevalence of diabetic kidney disease, it is a leading cause of kidney failure in the western world. There are few treatment options directly targeting the kidney.

The overall aim of this thesis was to dig past the current state of knowledge and delve deeper into the proteomic profile of diabetic kidney disease. By the use of discovery based and targeted mass spectrometry, the kidney proteomes of two diabetic mouse models reflecting type 1 and type 2 diabetes with early kidney disease were investigated and the serum proteomes of diabetes patients with various degrees of albuminuria were compared.

In summary, the majority of the changes were seen for proteins with expression in the tubules, the reabsorbing organ in the kidneys and not in the glomeruli, which is the filtering unit. The findings in this thesis could imply that we need to re-think the target of kidney medication and the importance of tubuloglomerular feedback.

For more information, please contact Peter James.
   

Ola Kilsgård: Proteomic profiling of bacterial host adaptation: Racing the Red Queen

(2016)

Despite the discovery of antibiotics almost a century ago, infectious diseases continue to be a substantial cause of human mortality and morbidity worldwide, especially in developing countries. The adverse affects of infectious diseases are thought to increase over the coming years as the widespread misuse of antibiotic leads to the emergence of strains for which current therapies are ineffective. The last decades has also seen a large increase of animal pathogens crossing the species barrier to cause disease in humans. To be able to reverse these negative trends we need better knowledge of the events leading to the adaptation of these pathogens to their host. This thesis aspires to increase our understanding of bacterial host adaptation with the hope of finding new targets for diagnostic and therapeutic treatments. In this thesis the development and application of novel mass spectrometry based methods for investigating bacterial host adaptation is studied. The developed methods are based on state of the art mass spectrometry proteomics, which allows the identification and quantification of in principal any expressed protein from a biological sample. The power of this analysis method was used to simultaneously quantify sets of bacterial and host proteins with a specific role in the infection course. These protein measurements are then used as standardization curves to obtain and account for any variation between biological states. The developed methods are combined to construct a quantitative model, depicting host – pathogen interactions and changes during infection progression. The model was used to determine the degree of host adaptation resulting of sequential passaging of the human pathogen Streptococcus pyogenes in a mouse infection model. In summery, this thesis has increased out understanding of the complex interactions leading to host adaptation of bacterial pathogens by the development of a quantitative model for bacterial infections. In addition, this thesis suggests a new approach for biomarker discovery and validation, by using standardization curves of potential biomarkers. The research conducted in this thesis has the potential to lead to increased clinical diagnostic and treatment opportunities of infectious diseases.

For more information, please contact Johan Malmström.
  

Payam Delfani: Biomarker Discovery in autoimmune diseases using antibody microarrays – Targeting systemic lupus erythematosus

(2016)

Autoimmune diseases affect millions of people around the world, and patients often face a lifetime of illness and treatment. Moreover, autoimmune diseases impose a heavy medical, financial and emotional burden on patients, patient’s families, and society. This PhD thesis addresses one of the core autoimmune diseases called Systemic Lupus Erythematosus (SLE). SLE is a heterogeneous and complex disease, why it often poses a significant challenge for both clinicians and investigators. The current diagnostic, prognostic, classification and therapeutic approaches are associated with major bottlenecks, representing key unmet clinical needs. Currently, the performance of available laboratory assays is sub-optimal, and the diagnosis of SLE is largely established based on clinical judgment, after excluding alternative diagnoses.

In order to address the current unmet clinical needs outlined above, a translational research methodology was adopted using a re-optimized recombinant antibody microarray technology platform. This thesis, based on four original papers, revolves around two major topics, one focusing on evaluation and further development of the recombinant antibody microarray platform (Paper I), and one focusing on applying the microarray platform for identifying panel(s) of SLE-associated biomarker candidates through discovery studies (Papers II-IV).

In Paper I, the third generation of our in-house designed recombinant antibody microarray technology platform is presented. The aim of the study was to address a set of key technological features of the technology platform and bioinformatics-related topics, resulting in a state-of-the-art protein expression profiling platform. In Paper II, the aim of the study was to profile proteins in sera of a large cohort of SLE patients (including three different subsets) and normal controls to identify candidate biomarker signatures capable of discriminating SLE patients from healthy individuals. The array data showed that several multiplexed panels of SLE-associated biomarker signatures could be pinpointed. In Paper III, the ambition of the study was to further investigate whether serum biomarker signatures reflecting disease activity could be de-convoluted. The results showed that condensed serum biomarker signatures capable of classifying both inactive and active SLE patients from healthy controls and from each other could be delineated. In Paper IV, our microarray platform was utilized to investigate whether immunoprofiles reflecting apoptosis and disease activity events could be discovered in patients with SLE. The data showed that several panels of SLE-associated serum biomarker candidates could be decoded, in particular reflecting disease activity, but potentially also reflecting apoptosis processes.

In summary, the long term goal of this thesis has been to set the stage for the next generation of clinically useful tools, providing the clinicians with new, actionable information. Thus paving the way for improved diagnostic sensitivity and accuracy of classification, and ultimately targeted treatment strategy. This ambition was pursued by two lines of projects, one focusing on evaluation and further development of our antibody microarray technology platform (Paper I), and one focusing on applying the microarray platform for immunoprofiling of SLE patients (Papers II-IV).

For more information, please contact Christer Wingren.
  

Johan Teleman: Quantitative analysis of mass spectrometry proteomics data - software for improved life science

(2016)

The rapid advances in life science, including the sequencing of the human genome and numerous other techiques, has given an extraordinary ability to aquire data on biological systems and human disease. Even so, drug development costs are higher than ever, while the rate of new approved treatments is historically low. A potential explanation to this discrepancy might be the difficulty of understanding the biology underlying the acquired data; the difficulty to refine the data to useful knowledge through interpretation. In this thesis the refinement of the complex data from mass spectrometry proteomics is studied. A number of new algorithms and programs are presented and demonstrated to provide increased analytical ability over previously suggested alternatives. With the higher goal of increasing the mass spectrometry laboratory scientific output, pragmatic studies were also performed, to create new set on compression algorithms for reduced storage requirement of mass spectrometry data, and also to characterize instrument stability. The final components of this thesis are the discussion of the technical and instrumental weaknesses associated with the currently employed mass spectrometry proteomics methodology, and the discussion of current lacking academical software quality and the reasons thereof. As a whole, the primary algorithms, the enabling technology, and the weakness discussions all aim to improve the current capability to perform mass spectrometry proteomics. As this technology is crucial to understand the main functional components of biology, proteins, this quest should allow better and higher quality life science data, and ultimately increase the chances of developing new treatments or diagnostics.

For more information , please contact Fredrik Levander.
   

Venera Kuci Emruli: A molecular dissection of mantle cell lymphoma. From gene expression analysis to functional evaluation of selected targets

(2016)

Mantle cell lymphoma (MCL) is an aggressive form of B-cell lymphoma (BCL) with poor clinical outcome. In this thesis, which is based upon five original papers, different approaches have been used to pinpoint and investigate molecular events that promote MCL tumor growth, progression or resistance to treatment. The aim has been to identify novel targets with the potential to eradicate malignant cells, including more resistant cell clones.

In Paper I-III, a previously defined MCL specific disease marker, SOX11, was functionally studied. Our results demonstrate that SOX11 is a tumor suppressor-like gene, exhibiting a central role in MCL tumor growth control. Using gene expression studies, we identified HIG-2 and CD24 as important downstream targets of SOX11, and due to their cell surface localization on MCL cells they are considered of potential therapeutic interest. Moreover, using functional screening with substances targeting components of the WNT pathway, WNT-related targets with SOX11-dependent growth regulation were identified. One of the targets, V-ATPase, a pH regulator, was shown to be sensitive to SOX11 levels. Validation studies confirmed that V-ATPase is localized on the plasma membrane of MCL cells, and is thus an interesting candidate for antibody-based treatment.

Despite the high response rate of MCL patients to initial treatment, a large number of patients relapse with a more treatment resistant disease. In Paper IV, the molecular mechanism behind developed resistance to cytarabine, a commonly used chemotherapeutic in treatment of MCL was investigated. Our results show that resistance to cytarabine in MCL is mediated by down-regulation of dCK at protein level. Importantly, cytarabine resistant cells were also shown to exhibit cross-resistance to other chemotherapeutic drugs (nucleoside analogues) acting similarly to cytarabine. Considering these findings, patients demonstrating cytarabine resistance in clinic should be offered non-nucleoside analogue based treatment.

Finally, in Paper V, we investigated the presence of a rare population of more treatment resistant cells, identified as side population (SP), in MCL. SP cells isolated from MCL patient material exhibited up-regulated expression levels of the early cell activation markers CD69 and CD44, in agreement with a stem-cell like origin. Of major importance, an enrichment of SP cells was observed in chemotherapy exposed cells, indicating that SP cells have functionally important features, and need to be further investigated in MCL.

For more information, please contact Sara Ek.
   

Anna Säll: Recombinant antibodies – Tools for building proteomic platforms

(2015)

This thesis, which is based on four original papers, covers different aspects of the evolvement and development of proteomic platforms and the construction of human synthetic antibody libraries for the isolation of antibody reagents has been very central. There is an accelerating need for technologies capable of providing proteomic analysis of high sensitivity with good accuracy and an overall broad coverage as well as for biomarker validation. Paper I, focused on the design and construction of human synthetic antibody libraries for the isolation of scFv antibodies suitable for the use in different affinity proteomic platforms. High-throughput procedures for the isolation as well as screening of antibodies was established and provides a pipeline capable of selecting antibodies against a large number of target antigens in parallel. Furthermore, scFv antibodies generated from these antibody libraries were validated for the use in antibody microarrays and were also utilized in various affinity-based proteomic platforms. In paper II and III, two different approaches are taken to address problems associated with proteomic analysis. Technologies capable of biomarker validation are highly required for increasing the implementation of protein biomarkers in clinical use. A method combining affinity enrichment with the use of scFv antibodies and mass spectrometric read-out were developed in paper II. The platform allows multiplexed detection of low abundant target proteins in human serum samples. Paper III focused on establishing a platform to facilitate the detection and investigation of bacterial virulence factors by using Streptococcus pyogenes as a model system. Bacterial infections are still a serious health problem around the world and analysis exploring bacterial strategies promoting survival and proliferation within the human host are of great importance. The performance of an affinity-based platform is dependent on the functionality of the used affinity reagents. In paper IV, a semi-targeted platform for biomarker discovery were enhanced by taking advantage of an oriented antibody immobilization strategy. In conclusion, this thesis presents a pipeline for development of human scFv antibodies suitable for proteomic analysis as well as the establishment and evolvement of various proteomic platforms.

For more information, please contact Carl Borrebaeck.
    

Frida Rydnert: Circulating human basophils and dendritic cell subsets. Aspects of antigen recognition and presentation in type I allergic immune responses

(2015)

Allergic diseases currently affect a third of the population in the industrialized parts of the world. Despite this, the cellular and molecular mechanisms involved in the induction and activation of allergic diseases are not fully clarified.

Dendritic cells (DCs) link the innate and adaptive immunity and play vital roles in protective immunity against pathogens, as well as in inducing tolerance to self-antigens, by regulating T cell responses. In allergic reactions, a dysregulated immune response is mounted against harmless compounds, such as pollen. Basophils are mainly known as effector cells in allergic responses where they release e.g. histamine after allergen-recognition, but basophils are also thought to be involved in other human disorders such as parasite infections and organ rejection, and were recently proposed to act as allergen-presenting cells in mice. This thesis, based upon five original papers, describes the role of DCs and basophils in allergy and immune regulation and give insights into mechanisms underlying allergic responses. In Paper I, we demonstrate a potent ability of human CD1c+ DCs, in contrast to plasmacytoid DCs and basophils, to activate Th2 responses in vitro, upon grass pollen stimulation of cells from allergic individuals. By performing phenotypical profiling of multiple pattern recognition receptors (PRRs) on four DC subsets, as well as basophils, isolated from human peripheral blood from allergic and non-allergic individuals, we demonstrate discriminative PRR expression profiles of the DC subsets and basophils in Paper II and IV, respectively. In Paper III and IV, we describe how the cellular frequencies of DC subsets and basophils, as well as their expression levels of PRRs and Fc receptors, alter during the first year of allergen-specific immunotherapy (AIT), proposing a modified ability of DC subsets and basophils to interact with allergens. Furthermore, in Paper V, we designed and produced a hypoallergen and used the basophil activation test to confirm its hypoallergenicity. This hypoallergen could potentially be used in AIT, instead of allergen extracts, to decrease the risk of adverse systemic reactions.

In conclusion, we have identified subset-specific characteristics within the DC population in both function and phenotype, implying subset-specific responsiveness when targeted for treatment of immunological disorders such as allergy. We further propose new ways in which basophils interact with their surroundings.

For more information, please contact Malin Lindstedt.
   

Ufuk Kirik: Development of computational methods for cancer research: Strategies for closing the feedback loop in omics workflows

(2015)

As the ultimate workhorses of the living things, proteins undergo significant regulatory activity throughout the lifetime of a cell or an organism. Many complex diseases effect the protein composition, expression or modification in the cells or tissues they arise in. It is then no surprise that proteomics is a field full of promise, one which is expected to generate significant insights towards functional characterization of cancer and deliver potential targets of diagnostic, prognostic or therapeutic value. It is also a science in its teens, which still grows and keeps changing as it grows with the technological advances in instrumentation as the driving force.Since data analysis routines are yet to be established fully, functional characterization of protein expression regulation in cancer remains an open question. The work presented in this thesis provides an overview of the field based on technological, computational and biological aspects in the introductory chapters, introduces a novel method for functional evaluation of changes in protein expression and demonstrates its utility in PAPER I, and describes the insights gained from investigating proteomes of several different types of human malignancies.PAPER I underlines the challenges in functional analysis of expression data, especially from LC-MS/MS experiments, and describes a method based on a relatively simple mathematical model, which is used for subsequent analyses. PAPER II describes a study on soft-tissue sarcomas, with the proteomic analysis revealing insights to protein expression patterns potential differentiation paths. PAPER III demonstrates a pairwise comparison of malignancies of gastroesophageal track and corresponding normal tissue; we highlight several proteins and pathways as likely targets of expression regulation. PAPER IV and V on the other hand focus on breast cancer. PAPER IV presents results from an investigation of immortalized breast cancer cell lines and raises the question of how well these model systems represents the tumours they are expected to be alike. PAPER V demonstrates the therapeutic potential of inhibiting oestrogen signalling in ER+ breast cancer, using a luminal type patient-derived xenograft mouse model.Collectively, this thesis presents some of the key concepts in quantitative proteomics workflows, elaborates on the importance of data processing routines and through the papers in the appendix demonstrates the potential of functional analysis algorithms in generating insights to cancer biology.

For more information, please contact Peter James.
   

Marianne Sandin: Quality control for high-throughput quantitative proteomics - Harnessing the potential of label-free LC-MS

(2014)

Multiplex protein quantification, proteomics, is essential for uncovering new biomarkers and understanding biology. Liquid chromatography coupled to mass spectrometry (LC-MS) is the predominant technique for these measurements. In this thesis, optimization and quality control strategies for different phases of the LC-MS pipeline have been developed. 

Due to the inherent complexity and dynamic range of the proteome, pre-fractionation is commonly employed in a general LC-MS setting to uncover low-abundant proteins. In paper I, we performed a (qualitative) comparative study of the proteins identified in a shotgun setup. It was found that SDS-PAGE, coupled to tryptic digestion, had the largest yield.

In paper II, we introduced a quantitative quality control method on peptide level for the relatively computationally demanding, but experimentally high-throughput, label-free LC-MS workflow, where we discovered both considerable differences as well as complementary properties between software solutions. 

The two integral parts of label-free data processing, feature detection (peptide quantification) and alignment (peptide identity propagation between samples), were further examined in papers III and IV. The complementary quality of software modules discovered in paper II was utilized in paper III, where we showed that a combination of different feature detection methods results in higher peptide coverage beneficial for downstream statistical inference. 

Since the establishment of proteomics as a high-throughput science, large-scale bioinformatics has become necessity. In paper IV, an alignment algorithm was developed where parameters are estimated on the fly from underlying data, an important step towards avoiding turning data processing into the future bottleneck of proteomics.

Finally, in paper V, we performed an extensive evaluation of the relative potential of discovery and validation LC-MS (shotgun and SRM), based on the method introduced in paper II. Extensive data handling optimization was performed from normalization to quantification and statistical implications were assessed. 

In summary, we have shown that large-scale label-free LC-MS can be combined with equally high-throughput quality control to create a competitive option in the hunt for biomarkers.

For more information, please contact Fredrik Levander.

Frida Pauly: Biomarker discovery in cancer using affinity proteomics - Studies of B-cell lymphomas and breast cancer

(2014)

The continual increase in incidence of cancer has created an urgent need for improved methods for diagnosis, prognosis and/or treatment. In this context, the field of biomarker research has expanded immensely during the last few years. This thesis, based on four original papers, revolves around protein biomarker discovery in cancer. For this purpose, an in-house design recombinant antibody microarray technology was used, a proteomic technology which has in the recent years been established as a sensitive, high-throughput method for protein expression profiling of complex samples. 

In paper I, we presented the first array setup for analysis of formalin-fixed, paraffin-embedded (FFPE) tissue, the traditional method for handling and storing tissue material in clinics, using recombinant antibody microarrays and thus identified an attractive way of addressing the extensive repertoire of retrospective FFPE samples available worldwide. 

B-cell lymphoma (BCL) is the most common type of hematological malignancy, but diagnosis and prognosis is inherently difficult to establish due to the large heterogeneity of the disease. In paper II, we confirmed the heterogeneity on a plasma protein level for four different BCLs. Moreover, immunoprofiles dividing these BCLs into subgroups were identified, and the subgrouping of one, diffuse large B-cell lymphoma (DLBCL), was correlated with survival. In paper III, the immunoprofiles of DLBCL patients undergoing treatment were investigated, revealing signatures which distinguished DLBCL patients from healthy controls, and moreover, delineated immunoprofiles associated with prognostic measures. In addition, the subgrouping of DLBCL found in paper II was pre-validated, and potential prognostic markers which, in this patient cohort, improved the prediction power of today’s golden standard for prognosis, the International Prognostic Index, were identified. 

Paper IV was aimed at identifying biomarkers associated with early breast cancer, the most common cancer type to afflict women, using the immune system as an early sensor for disease. In the study, we identified previously known and novel proteins associated with early breast cancer. In addition, anti-tumor and tumor promoting proteins implicated in the process of cancer imunnoediting were identified, demonstrating that a minimally invasive blood sample harbors disease-specific information reflecting early breast cancer and biological process thereof.

In summary, this thesis has contributed to the development of the recombinant antibody microarray technology by extending the range of sample formats available for analysis. In addition, immunoprofiles delineating cancer heterogeneity, effects of cancer treatment over time, and the effects on immunoprofiles in the early stages of cancer have been identified, which may potentially, in the long run, come to the benefit of cancer patients through improved diagnosis and prognosis.

For more information, please contact Christer Wingren.
    

Lena Nordström: SOX11 in mantle cell lymphoma - Novel tools for diagnostic, prognostic and functional investigations

(2014)

The goal of biomarker discovery is to identify disease defining molecules, which could be used to improve early detection and/or to predict survival and response to treatment. 

This thesis is about the transcription factor SOX11, a protein discovered in the context of mantle cell lymphoma in 2008, which shows great potential to be used as a clinical relevant biomarker. The aim of this thesis is to extend the use of SOX11, by providing novel tools for research applications as well as for diagnosing and stratifying mantle cell lymphoma patients. 

The results from my studies are presented in four papers in the thesis. First, we developed a monoclonal SOX11 antibody for the diagnostic classification of mantle cell lymphoma patients. Using this antibody, we showed that the expression of SOX11 correlates with patient survival and that inclusion of either SOX11 or p53 into the well-established prognostic index MIPI improve patient classification. To understand the growth regulatory role of SOX11, DNA binding sites and interacting protein partners needs to be identified. We continued to generate and assess the specificities of SOX11 targeting antibodies for immunoprecipitation assays, with the aim to get further insight into SOX11 signaling pathways using chromatin immunoprecipitation coupled to next generation sequencing or co-immunoprecipitation coupled to mass spectrometry. Finally, we investigated the epigenetic regulation of SOX11 and demonstrate the ability of re-expression using histone deacetylase inhibitors. 

In conclusion, this thesis presents novel tools, including the developed SOX11 antibody and the improved prognostic index for stratifying mantle cell lymphoma patients. Additionally, the developed SOX11 antibody has expanded our research applications to include flow cytometry and immunoprecipitation assays.

For more information please contact Sara Ek.
    

Linn Petersson: Tailoring of antibody arrays - Technical advances towards global proteome analysis

(2014)

he number of cancer and autoimmune disease diagnoses increases in our society, which place high demands on the health care system. Being able to tailor treatments for each individual patient is called "Personalized medicine", and could revolutionize the care in the future. This type of medicine requires biomarkers that could be used for diagnosis, prognosis and choice of therapy. In our group, we have developed an antibody microarray discovery tool that can analyze thousands of proteins in clinical samples in one single experiment. By identifying differences in protein expression profiles, biomarkers for various diseases can hopefully be found.

To date, antibody microarrays with an overall foot print of < 1 cm2, based on 18x103 µm2 (⌀ ~150 μm) sized spots at a density of ≤ 2,000 spots/cm2, have mainly been produced. Considering the size and complexity of the human proteome, this microarray design will not be able to harbor the number of antibodies required to perform global proteomics, demonstrating the need for novel, miniaturized high-density array designs. Increasing the number of antibodies on the arrays will generate a logistical problem when it comes to purification and immobilization of antibodies, which requires new technical solutions. The aim of this thesis was to develop new methods to produce the next generation of antibody arrays towards global proteome analysis. The first part of this thesis has involved design of miniaturized antibody arrays. The second part has been focused on developing antibodies with a photoreactive property, with the long-term goal to facilitate the logistics when it comes to purification and immobilization of antibodies.

In conclusion, new methods to fabricate miniaturized antibody arrays, focusing on both miniaturization and antibody immobilization, for large-scale protein analysis of clinical samples have been designed. In the future, this could lead to better discovery tools for identifying biomarkers for improved diagnosis, prognosis and choice of treatments for various diseases.

For more information, please contact Christer Wingren.

   

Anna S. Gerdtsson: Advances and applications of antibody arrays - Proteomic profiling of pancreatic disease

(2013)

Recombinant antibody microarrays have advanced into indispensable tools for large-scale, high-throughput multiplexed serum proteomics. This thesis, based upon five original papers, deals with the development of an in-house designed antibody microarray platform, and its applications for serum profiling of pancreatic disease.

Pancreatic cancer is the 4th deadliest cancer, with a 5-year survival rate of only 6%. In order to increase the survival of this deadly disease, novel diagnostic biomarkers for earlier detection will be essential. In paper I and II of this thesis, we identified candidate biomarker signatures for predicting pancreatic cancer among healthy controls and pancreatitis. Pancreatitis (pancreatic inflammation) is symptomatically highly similar to pancreatic cancer, and biomarkers able to discriminate pancreatic cancer from pancreatitis would be of great clinical value. Pancreatitis appears in mainly chronic, acute, and autoimmune manifestations, and like for pancreatic cancer, there is a lack of high-performing biomarkers for diagnosis and stratification. In paper III, we applied antibody microarrays for pancreatitis protein profiling, and presented tentative biomarker signatures for the three main subtypes of this disease.

In parallel to performing clinical applications of the antibody microarrays, technical efforts for improving and expanding the use of the platform have also been conducted. In paper IV, we studied the impact of the antibody-surface interplay, and evaluated different solid supports for antibody microarray production. We also took the first steps towards developing a user-friendly ELISA-like multiplexed biomarker assay, by presenting the first plate-based recombinant array-in-well set-up. In paper V, we designed protocols for an increased utility of the antibody microarray platform, to comprise not only targeting of proteins, but also serum/plasma profiling of glycan and carbonyl groups. Post-translational modification of proteins, like glycosylation and carbonylation (oxidation) is often altered in disease, and biomarkers based on differentiated levels of these modifications may complement traditional protein biomarkers. Proof-of-concept was demonstrated for preeclampsia, a common pregnancy disorder, for which the results indicated that particularly the level of carbonylation could be used for diagnosis and stratification.

In conclusion, the work in this thesis has contributed to an improved and increased utility of the recombinant antibody microarray technology, and demonstrated its use for serum proteomic profiling of pancreatic disease. 

For more information, please contact Christer Wingren.
   

Mattias Levin: Human IgE - What we can learn from repertoire studies and how we can use them as tools.

(2013)

Antibodies of the IgE isotype play a key role as the allergen recognizer in allergic disease, which today affects as much as a third of the population in the industrialized parts of the world. Despite this crucial role of IgE, our knowledge of IgE repertoires and how we can utilize the information hidden in monoclonal IgE to improve the quality of life of allergic patients, is limited. This thesis aims at describing our current understanding of human IgE repertoires and how we can use human monoclonal allergen-specific IgE as multitools in attempts to design improved diagnostic tools and novel therapeutic candidates.

The first part of the thesis, based on five original papers, discusses some fundamental features of human IgE repertoires and how such repertoires evolve in response to allergen, either via natural exposure or in the course of allergen-specific immunotherapy. Further, the involvement of IgE repertoires developed at local tissue sites, and how such repertoires may complicate diagnosis, is discussed.

In the second part, the focus is turned to human monoclonal IgE, and how we can learn useful lessons from studying the interactions between such antibodies and allergen. This part also describes the interaction between isolated allergen-specific monoclonal IgE and major allergens. Further, it discusses a range of potential applications for human monoclonal IgE, such as how we can use the information gained to design novel hypoallergenic variants to be used in allergen-specific immunotherapy.

In conclusion, this thesis outlines what we may learn from studies of human IgE and how we can use the information gained from such studies to improve the quality of life of allergic patients.

For more information, please contact Mats Ohlin.
   

Henrik Johansson: GARD - Genomic Allergen Rapid Detection. A testing strategy for assessment of chemical sensitizers

(2013)

The modern world is increasingly dependent of the use of chemicals. The chemical industries have greatly contributed to the high standard of living in industrialised societies, and chemical compounds surround us in everyday life. Unfortunately, a vast number of chemicals cause adverse effects on the environment and human health. One such concern is chemical hypersensitivity, which is a state caused by the human immune system. Upon exposure of certain chemical substances, the body will in some instances mount immunologic responses, giving rise to clinical symptoms such as irritation and damage on skin and impaired function of the respiratory tract.

In order to limit the usage of chemical compounds that induce hypersensitivity, often referred to as sensitizers, risk and hazard assessment of chemicals are crucial. Historically, toxicological hazard assessment has been performed using animal-based test methods. However, a number of factors, both political and ethical, make animal-based test methods for assessment of sensitizers unsuitable. Because of this, the development of alternative, animal-free test methods is very high priority research. However, no validated alternative is yet available that can singlehandedly replace the animal models used to date.

This thesis presents a novel in vitro assay for assessment of sensitization, called Genomic Allergen Rapid Detection – GARD. It describes the identification of a predictive genomic biomarker signature in MUTZ-3 cells, able to discriminate between cell samples stimulated with chemical skin sensitizers and non-sensitizers. These findings were used to formulate guidelines and protocols for the novel assay. The functionality and predictive performance of GARD have been demonstrated with results from predictions of blinded chemicals. In addition, GARD has been explored for the possibility to predict respiratory chemical sensitisers. The ability provide mechanistic information for two different endpoints makes GARD an attractive and unique assay for safety assessment of chemicals in an in vitro environment. In conclusion, this thesis discusses GARD in relation to trends in modern toxicology and demonstrates how accurate and safe assessment of chemical sensitizers can be performed without the use of animal experiments.

For more information, please contact Malin Lindstedt.
    

Paolo Cifani: Building a map of the breast cancer proteome - Strategies to increase coverage

(2013)

Amongst the various –omics sciences, proteomics has the highest potential for functional characterization and consequently can contribute significantly to the field of cancer research. In particular, the focus of this thesis is on breast cancer. Alas, since state-of-the-art technologies cannot meet the complexity of upper eukaryotic proteomes, a complete resolution of clinical samples is still unachievable. Comprehensive mapping of proteins involved in cancer and of their PTMs is proposed in this thesis as a general strategy to increase the output of mass-spectrometry based proteomics. Different approaches to improve the coverage of this map are proposed: optimization of sample fractionation, focusing on difficult sub-proteomes, targeting of specific biological processes and optimization of data analysis. A combination of these approaches will provide a growing collection of empirical MS-spectra, which will enhance the detection by shotgun proteomics and facilitate the transition towards the development of targeted assays.

For more information please contact Peter James.
    

Malin Nordström: Biomarker discovery in cancer and autoimmunity using an affinity proteomics platform - a tool for personalized medicine.

(2013)

Identification of novel biomarkers for early diagnosis, prognosis, and treatment decision will be instrumental for improving disease outcome for patients suffering from complex diseases, such as cancer and autoimmune disorders. Within personalized medicine, the use of circulating biomarkers will allow clinicians to tailor medical treatment based on individual characteristics of each patient, using minimally invasive sampling.

Clinical biomarker discovery studies are faced with a number of challenges, including access to large cohorts of well-characterized samples and high-throughput analysis platforms that can target numerous proteins simultaneously, while using minute volumes of sample. To meet these demands, affinity proteomics have recently been evolved as a key platform for biomarker discovery.

The aim of this thesis was to further optimize selected key features of our affinity proteomics platform, recombinant antibody microarrays, and to apply the platform in clinical studies. In the optimization studies I have i) evaluated and further developed the on-chip stability of antibodies included on the arrays, and ii) expanded and optimized the analysis protocol to now also enable targeting of urinary proteins.

In the two application studies, I have demonstrated the feasibility of using antibody microarrays for identification of protein biomarker signatures in both cancer and auto-immunity. First, by analyzing urine and serum samples from patients with systemic lupus erythematosus, I have identified candidate biomarker signatures for monitoring of disease activity and renal involvement. Second, I have identified plasma protein patterns that potentially could be used for stratification of heterogeneous patient groups into sub-groups of high or low risk of having prostate cancer.

For further information, please contact Christer Wingren.
   

Sissela Broos: Specific targeting of antigen-presenting cells for immunotherapy

(2013)

Immunotherapy, which involves strategies to activate, enhance or redirect immune responses to achieve long-lasting immunity, has the potential to play an important role in the treatment of a variety of diseases. This thesis, based on four original papers, highlights different approaches to modulate immune responses by specific targeting of antigen-presenting cells.

The first two studies focuses on regulation of allergic responses. In Paper I, we showed that biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles influence allergen-specific T cell activation, suggesting a beneficial role as adjuvants in allergen-specific immunotherapy. In Paper II, the histamine H4 receptor was shown to mediate histamine-induced activation of human dendritic cells and enhance their Th2 cell stimulating capacity in response to allergen. This suggests that blocking of the histamine H4 receptor may be a promising strategy to modulate allergic responses.

The last two studies involve strategies to optimise CD40-targeted cancer immunotherapy. In Paper III, adsorption of agonistic anti-CD40 monoclonal antibodies to γ-PGA nanoparticles was shown to enhance their efficacy and reduce the systemic release of cytokines after local drug delivery. In Paper IV, we demonstrated that increasing the affinity and the isoelectric point of an anti-CD40 antibody resulted in enhanced potency and tumour retention of the locally applied drug. The above strategies may thus serve to improve the efficacy and safety of local CD40-targeted immunotherapy.

In conclusion, this thesis underlines the promise of directing immunotherapies to antigen-presenting cells, and in particular dendritic cells, which are central decision makers of the immune system.

For more information, please contact Malin Lindstedt.
     

Linn Antberg: A proteomic dissecton of breast cancer. Via cells and organelles to pathways

(2013)

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. Breast tumours are extremely heterogeneous, and each patient’s disease has different causes, prognosis and appropriate treatments associated with it. To enable a more individualised therapy, there is a substantial need for better characterisation of tumours and a deeper understanding of the molecular mechanisms of disease initiation, progression and treatment resistance.

During the last decade, shotgun proteomics has evolved as a powerful tool, complementing the fields of genomics and transcriptomics, for the elucidation of pathway and network changes in cancer. The proteome is, however, with its large dynamic range and numerous post-translational modifications, tremendously more complex than the genome and an additional level of complexity is provided by the spatial arrangements of proteins in different organelles.

In this thesis we have addressed technical challenges such as optimising proteomic coverage by evaluating different protein and peptide separation methods and mapping proteins to organelles with density gradient organelle fractionation followed by proteomic quantification of the fractions. Further, we applied both a discovery mode of proteomics and a focussed pathway centric method to evaluate the response of breast cancer cell lines to chemotherapeutic agents. Proteomic pathway analysis was also utilised to investigate and explain the differential response to therapy in different breast tumour subpopulations.

For more information, please contact Peter James.

Sandra Sernbo: Functional studies of SOX11 and T-STAR. Translation of tumor-associated genes into clinical and biological insight

(2013)

Cancer is a disease that will affect most of us during a lifetime. Although the survival of cancer patients has incresed due to better treatment and earlier diagnosis, the prevalance of the disease is still increasing. This increase in the number of cancer cases diagnosed will impose a challenge to the cancer care and higher costs for socitey. Personalized medicine aims at reducing these costs while at the same time improving the care for the patient, mainly by using a more individualized therapy, but also a more preventive cancer care and even earlier diagnosis. For example, a more indivivualized treatment can target specific genetic aberrations found in a patient, and avoid treating those patients who lack the aberrations and therefore will not respond. To accomplish the goals of personalized medicine, biomarkers that can divide patients into different subgroups are needed. 

The aim of this thesis was to explore SOX11 and T-STAR as potential biomarkers in cancer. I was also interested in the function of these proteins in the tumor tissues where they are specifically expressed. Immunohistochemistry on tissue microarrays was used to evaluate the expression of SOX11 and T-STAR in primary tumor tissues, and the expression was then correlated to survival in the patients. The function of the proteins was explored using RNA interference and overexpression experiements in cancer cell lines.

The results from my studies are presented in five papers in this thesis. First, we could confirm the diagnostic role of SOX11 in mantle cell lymphoma (MCL). Second, I show the prognostic potential of this protein, as patients with high expression of SOX11 are associated with a better survival, both in MCL and high-grade epithelial ovarian cancer. In MCL, SOX11 could possibly complement the MCL international prognostic index MIPI, a prognostic biomarker already in use in this disease. Third, in both tumor types SOX11 functions as a tumor-suppressor in vitro. Regarding T-STAR, its expression is correlated with better outcome for breast cancer patients, and therefore shows potential as a prognostic biomarker. Our studies also show that T-STAR has a growth-inhibitory function in breast cancer cell lines.

In conclusion, both SOX11 and T-STAR are biomarkers that could be used in personalized medicine to subgroup patients and potentially guide treatment decisions. Both proteins function as growth-inhibitors in vitro, which correlates to the better prognosis for the patients with tumors expressing the proteins in vivo.

For more information, please contact Sara Ek.
   

Laura von Schantz: Engineering protein-carbohydrate interactions - lessons from natural and evolved carbohydrate binding modules

(2012)

Protein-carbohydrate interactions are of central role in all living organism. In the studies presented in this thesis several strategies for engineering such interactions have been investigated. In the first study the binding affinity of a carbohydrate binding module (CBM) was improved a 100-fold through affinity maturation. Also, in that study it was discovered that all the selected, matured mutants carried a mutation in common (glutamic acid), which was shown to be responsible for the increased affinity.
Furthermore, the binding interaction of two different CBM was analyzed with x-ray crystallography. One of the CBM can only bind xylans, while the other binds also beta-glucans and xyloglucan. The crystal structures revealed that the binding cleft of the multi-specific CBM is flexible, permitting reorientation of side-chains to avoid steric clashes. Also the multi-specific CBM harbored an important phenylalanine that due to its chemical composition has a pi-electron surface and can interact with hydrogen atoms on the different ligands it recognizes. The specific CBM on the other hand harbor a leucine in this position and can only interact with xylans. In the last study, the capacity of CBM for use as analytical tools in the characterization of the distribution pattern of cellulose derivatives was investigated. The study showed that some CBM are able to distinguish between cellulose derivatives with different substitution distribution. This is a potentially new application for CBM. In all, the thesis demonstrates strategies in which carbohydrate binding proteins can be generated, improved and utilized.

For more information, please contact Mats Ohlin.
    

Niclas Olsson: Global Proteome Survey -Transforming antibody-based affinity proteomics into a global discovery platform

(2012)

Proteomics is expected to generate new insights into biological processes as well as identify novel biomarkers and therapeutic targets since most biological functions are transmitted through proteins. However, due to the complexity displayed by a proteome and inherent limitations associated with current methodologies, proteomic analyses often result in incomplete coverage and inconsistent measurements. Clearly, the development of novel high-performing proteomic platforms will be essential in order to successfully decipher the human proteome(s).

This thesis, based on four original papers denoted I to IV, describes the development and applicability of a novel proteomic technology platform entitled Global Proteome Survey (GPS) capable of transforming affinity proteomics into a global discovery engine. The GPS methodology combines the best features of affinity proteomics and mass spectrometry, and is based on using antibodies specific for short C-terminal amino acid peptide motifs shared by many proteins. This opens up the possibility to identify and quantify significant portions of a proteome, while still using a limited set of binders in a specie independent manner (Paper I-IV). Furthermore, structural models were generated for a large set of the experimentally verified captured peptides and the matching antibodies (Paper III). The data generated novel insights into antibody-peptide interactions and showed that a few key residues were essential for establishing specificity and acting as anchor residues.

The GPS assay reproducibility was extensively tested (Paper I, II, and IV) and displayed median coefficient of variations in the range of 10-20% in tissue profiling. In addition, the sensitivity was demonstrated by successfully targeting proteins present in a range of abundance values spanning over a million down to less than 50 copies per yeast cell (Paper I and II).

In Paper IV, the first clinical proteomic application of GPS was demonstrated by generating in-depth proteomic insights of 52 breast tumor tissues. While using only 9 antibodies, the GPS-platform enabled identification and quantification of over 1300 proteins, and most importantly established a link between a molecular signature and tumor progression. Highly relevant and promising cancer-associated protein signatures related to histologic grade, estrogen receptor, and HER2/neu-status were identified.

In conclusion, we have developed and demonstrated the quantitative capability, reproducibility, sensitivity, and coverage of the GPS methodology. It provides important methodological solutions to the complexity of proteome analysis and may act as a valuable tool for analyzing large numbers of clinical samples in an accurate, sensitive, and discovery-based manner. Hence, antibody-based affinity proteomics have been transformed into a global discovery platform and will pave the way for novel proteomic insights into complex molecular pathways in health and disease.

For more information, please contact Christer Wingren.
 

Fredrika Carlsson: Antibody development - Examples from viral and proteomic studies.

(2012)

This thesis, which is based on four original papers, covers different aspects of antibody development. The overall goal of my work has been to obtain an increased understanding of antibody repertoires as they develop in vivo and in vitro.

In Paper I-III antibody repertoires was evaluated and used to dissect virus-induced antibody responses and characterize such antibodies. Furthermore, tools for more efficient studies of such characterized antibodies were developed. The study of antibody repertoires in this thesis reveals, not only insights of the composition of antibodies induced by a common virus, but also features for such binders developed by combinatorial and non-combinatorial approaches. Such developed antibodies may contribute with data important for future design of vaccine candidates.

In Paper IV, limitations in biomarker verification processes are addressed. A proof-of-concept study was developed, describing an approach with high-throughput potential relying on antibody fragment enrichment of proteins in complex serum samples followed by an MRM mass spectrometry readout. Taken together, the in vitro developed binders in combination with MRM readout provides a powerful method to quantify protein biomarkers in a high-throughput manner approaching the criteria for clinical usage.

For more information, please contact Mats Ohlin

Elin Gustavsson: SOX11 as a cancer biomarker - Expression and regulatory studies in B-cell lymphomas and ovarian cancer

(2010)

It is estimated that one third of all people in Sweden will be diagnosed with cancer during their lifetime. The outcome for a specific patient is related to early and correct diagnosis, and prediction of effective treatment regimens. Biomarkers are molecules that can aid the clinician in such decisions regarding diagnosis, prognosis and therapy. In this thesis, based on five original papers, the role of SOX11 as a novel diagnostic biomarker in MCL and prognostic biomarker in ovarian cancer is presented. In addition, it is demonstrated how antibodies against such cancer biomarkers, including SOX11, are generated and used in various applications. Expression of SOX11 in MCL is herein shown to be independent of Cyclin D1 and able to distinguish MCL from common morphological simulators. Furthermore, the prognostic relevance of SOX11 also for non-haematological disease is confirmed as expression of SOX11 in high-grade ovarian cancer relates to survival. The functional role for SOX11 is further demonstrated for expressing malignancies, as it appears to be a negative regulator of cellular growth in vitro, possibly by inducing expression of tumour suppressors. An example of the underlying regulatory mechanism for SOX11 expression in cancer is provided as the methylation status of its CpG promoter correlates to expression in lymphomas and ovarian cancers. DNA hyper methylation commonly represses genes in neoplastic cells and has the potential to be detected in blood as a diagnostic test of methylated malignancies.

For more information, please contact Sara Ek.

Anders Carlsson: Molecular serum portraits - A step towards personalized medicine

(2010)

Antibody microarray technology has the potential for playing a large roll in identifying serological biomarker panels for personalized medicine. The aim of this thesis, based on four original papers, was to investigate if information in the serum proteome could be extracted and used for diagnostic, classificational, prognostic or treatment predictive purposes in a range of diseases. In two studies (paper I and paper IV), the diagnosis and prognosis of breast cancer was addressed, also being the main focus of this thesis. In paper I, we identified a biomarker panel capable of stratifying serum samples from metastatic breast cancer patients from those of healthy controls. In paper IV, another panel, pre-validated in the same study, was deciphered that could be used to identify patients destined for metastatic disease in a group of newly diagnosed breast cancer patients. Paper II and III targeted immunotherapy of glioblastoma multiforme and the diagnosis and sub- stratification of two autoimmune diseases (SLE and SSc), respectively. Also in these cases, multiple biomarker panels were identified, each capable of separating predefined cohorts of patients with relevance for applications within personalized medicine. In conclusion, this thesis introduces the concept of personalized medicine; details the antibody microarray technology in general and the platform used for the experiments in paper I to IV; and describes the subsequent microarray data analysis.

For more information, please contact Christer Wingren.

Linda Dexlin Mellby: Design of antibody microarrays for global profiling of membrane proteins and soluble proteins

(2010)

Antibody-based microarrays have emerged as an established proteomic technology allowing multiplexed and sensitive profiling of complex proteomes, in a high-throughput and miniaturized manner. Recently, numerous applicative efforts have been pursued generating disease-associated protein signatures that now could be further explored for improved disease diagnostics, prognostics and classification.

The aim of this thesis, based on five original papers, was to develop the antibody microarray methodology even further, to allow for global proteome analysis. Accordingly, a miniaturization of the array features was established, allowing for high-density arrays to be fabricated. We showed that sensitive detection of protein analytes in pure system as well as complex serum samples could be achieved using this miniaturized recombinant antibody nanoarray set-up. In additional globalization efforts, we demonstrated that two, novel, recombinant antibody microarray set-ups, based on human scFv antibody fragments, could be designed for membrane protein profiling of intact cells and cell/tissue extracts, respectively. This will provide us with unique and novel means to delineate the membrane proteome, previously proven to be to be difficult to address using conventional proteomic approaches. Taking advantage of these technological developments in a follow-up applicative study, differentially expressed membrane proteins and water-soluble proteins were readily identified in preeclamptic placenta vs. normal placenta. The data showed that candidate disease-associated tissue protein signatures could be identified, that could help to decipher the complex features of preeclampsia at the molecular level. Finally, we demonstrated how a wide variety of protein analytes could be targeted in mantle cell lymphoma, adopting genome-based affinity proteomics, using a novel reverse-phase antibody microarray set-up. Altogether, these technological improvements will allow us to gain further insights into complex molecular pathways in health and disease

For more information, please contact Christer Wingren.

Ulrika Andréasson: Transcriptional analysis of cell-sorted B-cell lymphomas from high-throughput to focused identification of molecular targets

(2009)

B cell lymphomas (BCLs) are a group of severe cancers, afflicting both men and women at different ages. Gene expression profiling (GEP), have during the latest decades fundamentally contributed to a better classification and biological understanding of the different BCLs. Most studies using GEP have analyzed mRNA from tumor tissue constituted of both tumor cells and non-malignant bystander cells. This thesis is based on four original papers where pure, cell-sorted BCL cells are analyzed by GEP. This procedure allows identification of differences in tumor cells rather than variations due to cellular composition. These studies focus on identifying new prognostic, diagnostic and/or functional markers for BCLs, using four different approaches. In the first study, GEP of pure mantle cell lymphoma (MCL) cells were used to identify MCL-associated targets genes. Subsequent immunohistochemistry (IHC) using antibodies raised against a unique protein epitope signature (PrEST) was performed. Using this high-throughput strategy we were able to identify proteins either uniquely or highly expressed in MCL compared to normal lymphoid tissue. This study confirmed the usage of transcriptional screening to identify molecular tumor-associated targets, as well as suggest the PrEST-approach as a novel efficient technique to identify molecular targets with both a known and unknown identity. The indolent growing follicular lymphoma (FL) cells are dependent on interplay with cells in the microenvironment for survival. We analyzed, in a second study, pure FL cells for highly expressed genes encoding surface bound proteins. An aberrant expression of CX3CR1 on the surface, not only on FL cells but also on tumor cells in several other BCLs was identified. CX3CR1 is suggested to be involved in site-specific dissemination, the possibility to use CX3CR1 as a target for antibody-mediated intervention needs however further investigations. The indolent FL often transforms to diffuse large B-cell lymphoma (DLBCL-tr). Previous studies analyzing tumor tissue for markers involved in this high-grade transformation have revealed results with low concordance. In a, we analyzed purified FL and DLBCL-tr tumor cells and found that large inter-tumor heterogeneity still was observed. Despite the heterogenous gene expression, this third study identified distinct differences between tumor entities rather than differences due to various composition of bystander cell and two proteins, galectin-3 and NEK2 pinpointing a subgroup of DLBCL-tr over FL were identified. In the last study, we used a unique material of eight different B cell lymphomas and identified for the first time unique transcription factors for each of these highly purified entities. The identified TFs are not only potential new molecular targets but are also partly responsible for the differences in global gene expression in the different BCLs as demonstrated by unsupervised clustering. In conclusion, analyzing pure lymphoma cells, dramatically enhance the resolution of analyses and allowed for identification of tumor-cell related genes using different approaches. Downstream analyzes of these genes allow for identification of proteins with a major biological importance for BCLs and potentially of new biomarkers.

For more information, please contact Ulrika Andréasson

Kristina Lundberg: Subsets of human dendritic cells and their role in allergy

(2009)

The immune system has evolved to protect the human body against harmful invaders. At the same time, development of tolerance against self-derived and innocuous foreign antigens is of vital importance in order to avoid detrimental immune disorders. In allergic conditions, hypersensitivity is developed against harmless antigens, such as pollen. Dendritic cells (DCs) serve as a link between the innate and adaptive immune system and orchestrate the activity of T-cells. This thesis, based on four original papers, deals with distinct subtypes of DCs and their role in allergy, as well as mechanisms underlying DC regulation of ongoing allergic responses. By transcriptional profiling of DC subsets identified in blood and tonsils we demonstrated subset-selective gene expression, including many genes associated with immune responses. In addition, the resemblance between myeloid DC (mDC) subsets in tonsils suggests that they are highly susceptible to microenvironmental factors. Blood DC subsets were further investigated regarding characteristics associated with allergic responses and the data suggest a prominent role of one specific mDC subset in grass pollen allergy. Thus, functional studies of this subset may give important insights into the regulation of allergic responses in humans and identify novel targets for allergy therapy. In order to understand DC regulation of ongoing allergic responses, we assessed the influence of IgE antibodies as well as histamine and histamine receptor (HR) 1 and 4 on DC function and subsequent T-cell responses. Due to DC scarcity, these extensive functional studies were performed on DCs derived in vitro from monocytes. We demonstrated that HR4, similarly to HR1, mediated histamine-induced DC activation and HR4 on DCs also influenced T-cell activation in response to grass pollen allergen. Thus, HR4 may be involved in the maintenance of allergic responses and is a potential novel target in allergic conditions. Our assessment of DC modulation by IgE antibodies showed that IgE, in contrast to IgG1 and IgG4, augmented DC-induced allergic T-cell responses to grass pollen allergen. Thus, the production of IgE antibodies during allergic responses may result in a positive feedback loop via their influence on DCs. In conclusion, the work within this thesis has pinpointed subset-specific characteristics within the DC lineage, supporting individual roles in immune responses and further suggests that one specific myeloid DC subset has a prominent role in allergic rhinitis. Furthermore, mechanisms underlying DC-mediated perpetuation of allergic responses were identified. These insights may lead to development of novel allergy therapeutics which targets key mechanisms involved in DC regulation of allergic responses.

For more information, please contact Kristina Lundberg

Erika Gustafsson: Molecular evolution of a C5aR antagonist against inflammatory disease

(2009)

New anti-inflammatory drugs with fewer adverse effects than existing drugs may prove to be useful in the treatment of several inflammatory disorders. A critical step in acute inflammation is the infiltration of neutrophils into tissues. Therefore, molecules that target and inhibit this early inflammatory event are attractive to engineer to suit medical needs. The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formylated peptide receptor (FPR) and is therefore a potent inhibitor of activation and migration of neutrophils. However, as the majority of the human population has been exposed to S. aureus, pre-existing antibodies against CHIPS will be present in human sera. This thesis is based on four original papers, with the overall aim to decrease the interaction of CHIPS with pre-existing human IgG in order to tailor it for pharmaceutical purposes. Specific IgG epitopes on the CHIPS surface were mapped by the use of phage displayed random peptide libraries. This study showed that polyclonal CHIPS specific IgGs mainly recognize conformational epitopes exposed on the surface. In addition, amino acid residues in CHIPS that are involved in this interaction, as well as in C5aR inhibition were identified. Directed evolution is a process commonly used to improve certain protein properties without the need for detailed prior knowledge of the protein structure. This can be performed by the use of in vitro DNA recombination, a procedure by which beneficial mutations from a randomly mutated library can be recombined to generate new protein variants. In this thesis, the DNA recombination technology Fragment INduced Diversity (FIND), was applied in combination with molecular modeling and site-directed mutagenesis to generate CHIPS variants with low interaction with human IgG and retained ability to inhibit the C5aR. One CHIPS variant, designated ADC-1004, was selected for further studies. This new CHIPS variant, has high affinity for the C5aR and inhibits it efficiently, despite the truncation and seven mutations that mediate low interaction with pre-existing human IgG.

For more information, please contact Carl Borrebaeck

Jonas Persson: Selection and design in antibody phage display

(2008)

The immune system is an important part of the physiology of higher organisms. In the immune system antibodies play a key role in neutralizing and clearing potentially lethal threats from the environment. The importance of antibodies in the immune system has made them come to the attention as a tool in chemistry and medicine. This thesis, which is based on four original papers, deal with antibody development using phage display libraries. In the articles in this thesis efforts have been made to develop antibodies with the ability to distinguish between healthy cells and tumor cells with potential applications in diagnosis, prognosis and therapeutics. We have also made efforts in streamlining the process of extracting such antibodies from in vitro display libraries. In the first three papers, questions regarding antigenicity, antigen design, affinity maturation and specificity fine-tuning are highlighted and the importance of design of antibody libraries, antigens and methods of separation are key features of this thesis. Based on the first two papers in this thesis I conclude that using a simplistic design of the antigen may lead to an unsuccessful panning process of the chosen antibody library that when panned with a more efficiently designed antigen was found to contain antibodies of the desired specificity. This chain of issues leads to the development of several antibodies with differential recognition of and binding to the tumor-associated antigen MUC1. I conclude that these antibodies would be suitable candidates for use in cancer diagnosis and also have the possibility to be used in treatment. There is a great demand for affinity binders in proteomics and several efforts are in play to meet the demands for binders towards all constituents of the human proteome. We meet this demand in the fourth paper by developing a system for automatic wash and separation for use in high-throughput selections. This system is suitable of extraction of binders from phage display libraries against large numbers of targets.

For more information, please contact Mats Ohlin

Maria Jansson: Membrane protein proteomics - Novel method for membrane protein identification and quantification.

(2007)

Membrane proteins are fairly refractory to digestion especially by trypsin. Less specific proteases like elastase and pepsin are much more effective. However database searching using non-tryptic peptides is much less effective due to the lack of charge localisation at the N- and C-termini and the absence of sequence specificity. We describe a method for N-terminal specific labelling of peptides from non-tryptic digestions of membrane proteins which facilitates database searching and can be used for relative quantitation. The conditions for digestion using the non-specific enzyme Proteinase K have been optimised to obtain peptides of a suitable length for MS fragmentation. We show the effectiveness of the identification of membrane proteins using a plasma membrane preparation from a leukaemia cell line and demonstrate a large increase in the number of membrane proteins with small extra-membranar domains being identified in comparison to previous published methods.
Our method was then further developed for relative quantitation of membrane proteins. We describe this by using membrane preparations from Bacillus subtilis grown with and without the presents of 0.5% glucose and an isotopic labelling strategy. The results show good reproducibility of the calculated fold changes of the identified peptides within the same protein.
The method was then applied on plasma membrane proteins from cancer cells grown with and without the presence of oxygen. The aim was to find biomarkers for tissue hypoxia.

For more information, please contact Peter James

Kristina Larsson: Dendritic cells in allergy. In vitro models for studies of the human immune response.

(2007)

This thesis, which is based on four original papers, deals with human dendritic cells (DCs) in immunity and allergy. DCs play a key role in the regulation of immune responses by acting as the sentinels of the immune system, specialized in the uptake of a diverse range of antigens. These cells migrate to local lymph nodes and become highly efficient in inducing T cell proliferation, thus inducing adaptive immune responses. Therefore, DCs function as the link between innate and adaptive immune responses. Besides pathogenic microorganisms, the immune system encounters numerous proteins, many of which are harmless. A proper response against these harmless antigens is tolerance. The balance between induction of immunity or tolerance is in part determined by DCs. A dysfunction in the regulation of this balance may result in diseases such as allergy and autoimmunity.
The incidence of allergy has reached up to 40% of the children in industrialized countries and is increasing in developing countries. The reduced quality of life in these conditions increases the need to strive for commercial products of low allergenicity. Tests to verify these products are needed, and directives from the European Union aim for a reduction of animal tests and development of alternative in vitro models. A model based on human DCs would be suitable since they have a central role in the immune system and in allergy. To address this question we studied transcriptional profiles of monocyte-derived DCs (MoDCs) and the differentiated monocytic cell line MUTZ-3 induced by pro-inflammatory signals. We could demonstrate that the cell lines expressed a wide range of molecules that are important for DC function. Thus, MUTZ-3 closely resembles DCs and is suggested to serve as an in vitro model to predict allergenicty of novel compounds. Yet, the functionality of the cell line needs to be further evaluated. The effect of an allergen (timothy) on DCs was demonstrated by transcriptional profiling of grass pollen stimulated DCs from allergic and non-allergic individuals. Furthermore, memory effector T cells were demonstrated to have an instructive role in the cross-talk between DCs and T cells in both allergic and non-allergic conditions. This education of DCs was in part specific in allergy and supports the concept of DC-mediated sustained allergic inflammation in the periphery. In addition, the reported panel of genes reveal novel molecules involved in the enhanced inflammation. The genes reported specific for allergen stimulated DCs and in the subsequent DC/T cell crosstalk in allergy may be interesting targets in the search of novel drugs.
New methods for administration of vaccines are desirable to increase the speed and ease for delivery of vaccines in addition to improved safety. DCs are suitable targets for vaccine delivery since they are efficient inducers of adaptive immune responses. As they are located in mucosa and skin they can be reach by vaccine delivery to any of these tissues, e.g. via nasal administration. Nanospheres induce adaptive immune responses and function as both antigen carrier and adjuvants. Transcriptional profiling of MoDCs upon stimulation with nanospheres revealed activated pathways during uptake and processing of the antigen. Next, the efficiency of biodegradable nanospheres to induce DCs to mount adaptive immune responses needs to be identified.

For more information, please contact Carl Borrebaeck

Åsa Wåhlander: Chemical labelling methods for gel free based proteomics

(2007)

In this thesis different aspects of gel free procedures for mass spectrometry based proteome analysis are addressed, with emphasis on labelling techniques. It is based on three original articles where the effects imposed onto peptides upon modification are investigated, and the development of a new type of labelling strategy is described. Chemical modification has been of great importance for the peptide based proteomics approach, as they provide a way to impose desired properties onto the peptides. One possibility is to improve ionisation efficiency by introducing an easily protonated entity. By strategic placement of such a label, favoured ionisation of certain fragments upon MS/MS analysis can work to simplify the fragmentation spectra, and consequently facilitate their interpretation. In this work, such a charge-directing fragmentation label and its effects using a new type of mass spectrometer was investigated. One of the main applications for the various labelling techniques however, has been the ability to produce isotopic counterparts of peptides, which enables quantification of the sample constituents.  Several procedures to accomplish this, using a wide assortment of labels have, and are being developed. The Parent Ion Quantitation Scanning (PIQS) method described in this work, was developed aiming at more efficient global analysis of protein expression levels. The method was constructed to only sample peptides from differentially expressed proteins, thereby alleviating the large amounts of data usually generated, and only targeting relevant information, when aiming for analysis of differences in protein abundance.

For more information, please contact Peter James.

Helena Persson: Generation and evolution of human antibody repertoires

(2007)

This thesis, which is based on five original papers, covers different aspects of antibody specificity and diversity, with the overall goal to understand the makeup of antibody repertoires and their functionality as they occur in vivo or as they are developed in the laboratory.
Besides their importance in the adaptive immune response, antibodies have emerged as an invaluable tool in applications ranging from basic research to disease diagnostics and therapy. Antibody library technologies have during the past two decades re volutionized our ability to develop antibodies without the machinery of the i mmune system. However, in order to successfully create antibodies with desired binding properties a deep understanding of the antigen-antibody interaction is required.
Part of this work deals with different aspects of the molecular recognition mechanism of antibodies and how this knowledge can be used to construct antibody libraries with improved characteristics. In The first part of the thesis, I describe the development of a novel approach to create antibody libraries with increased functionality with respect to pre-defined groups of antigens. By constructing a library based on a hapten specific antibody sequence, a library highly enriched for binders to small antigens was cre ated. Import antly, we could show that this library was a superior source of hapten-binders compared to a l arger multi-purpose library. These kinds of focused librar ies offer an attractive alternative when conventional libraries fail to deliver useful binders.
Antibody library technologies have also allowed the investigation of antibody repertoires as they develop in vivo. In the second part of this thesis, we have studied the composition and specificity of antibodies of the IgE repertoire. This antibody isotype is of extensive medical importance as it is a key mediator of type I hypersensitivity or allergy. By using library and selection technologies it was possible to delineate the specificity of over 25% of the IgE-producing transcripts in a grass-pollen allergic individual. Apart from providing valuable insights into the pathogenesis of allergy, we have established a range of antibodies that can aid us in the quest to define ways through which human IgE antibodies recognize grass pollen allergens, which in turn can provide important clues in the design of new allergy vaccines.

For more information, please contact Mats Ohlin

Sofia Waldemarson: Profiling the cancer proteome. Studies on breast, ovarian and prostate cancer.

(2007)

Cancer is a complex and heterogeneous disease where cells have started to grow uncontrolled and this disease remains a major health problem. Because of diffuse symptoms it often presents at a late stage. Insufficient understanding of the different phenotypes hinders the development of targeted therapeutics and consequently patients show a diverse range of responses to a given treatment.  
This thesis is based on four original papers where mass spectrometry based proteomics has been used to study three different cancers; breast, ovarian and prostate cancer. Two-dimensional gel electrophoresis and shotgun proteomics LC-MS/MS based analysis has been applied.  
Ovarian cancers often have a bad prognosis because of late presentation. Diagnostic markers for early detection are urgently needed. Two studies in this thesis analysed ovarian cancer tumours for this purpose. Protein expression profiles from 2D-DIGE could separate the tumour subgroups and proteins differentially expressed with increased malignancy were identified. A more in-depth characterisation of benign and malignant samples was done using a shotgun proteomics approach and iTRAQ for quantitation.
Breast cancer consists of several pathological subtypes with different clinical presentations and outcomes. Using 2D-DIGE protein expression profiles were constructed for tumours previously analysed for gene expression to stratify these tumours. The subgroups found agree very well with groups found on transcriptional level and these correlate well with clinical information. Proteins characterising these subgroups could be useful as prognostic markers in the clinic.  
Somatostatin can potentially inhibit tumour growth in advance stage hormone refractory prostate cancer. A derivative of somatostatin that increases its half-life is interesting as a potential treatment. The effect of somatostatin and its derivative was studied in a prostate cancer cell line using 2DE. Differentially expressed proteins were identified. Somatostatin and its derivative were shown to exert the same effect on the cell line pointing at the derivate as a potential treatment.

For more information please contact: Peter James.

Lavinia Cicortas Gunnarsson: Novel binding specificities engineered into the scaffold of a carbohydrate binding module

(2007)

The growing field of biotechnology is in constant need of proteins that can function as recognition tools for separational, analytical and therapeutic purposes. Different molecular engineering approaches are applied on natural proteins in order to create variants with desired properties. This thesis is based on five original papers that deal with selection, characterisation and application of novel binding specificities engineered into the scaffold of a carbohydrate binding module that originates from a xylanase in the thermophilic bacterium Rhodothermus marinus. Molecular evolution studies on this scaffold allowed for the generation of variants that bind specifically to the carbohydrate targets xylan, Avicel?, mannan and xyloglucan. In addition, the scaffold employed in this work was also able to adopt specific protein recognition to a human IgG4 molecule. Apart from high binding specificities, the engineered proteins have additional properties such as high thermal stability and ease of production in Escherichia coli, which are advantageous in most applications. One of the papers in this thesis demonstrates the potential use of the created xylan-binding variants for detection of their target in wood fibres and plant sections. Also, generated variants with other binding specificities have the potential to find similar use as bioanalytical tools. In conclusion, the scaffold of the carbohydrate binding module engaged in the engineering studies of this thesis proved to be suitable for carrying diversity and has thus allowed for the creation of novel variants with diverse binding specificities useful in biotechnological applications.

For more information please contact: Mats Ohlin.

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Johan Ingvarsson: Design of recombinant antibody microarrays for global proteome analysis

(2006)

Antibody-based microarrays are among the new, rapidly emerging technologies within the field of proteomics that has the potential to evolve into a key proteomic research tool, providing novel means to perform early diagnostics, identify biomarkers and gain insights into disease biology.

The aim of this thesis, based on five original papers, was to develop antibody microarrays based on human recombinant scFv antibody fragments for large-scale comprehensive proteome analysis. In order to accomplish that, several of the key technological antibody microarray parameters critical for complex proteome analysis were carefully addressed and optimized (paper I-IV). By successfully optimizing each of the features, a state-of-the-art recombinant antibody microarray technology platform was successfully designed. The recombinant antibody microarrays could readily be used to perform highly sensitive and multiplexed protein expression analysis of complex samples, such as human plasma. In the final study (paper V), we applied the optimized microarrays to analyze pancreatic cancer serum samples and compared the protein expression signature to healthy controls. The results showed that a pancreatic cancer associated protein signature could be identified that could discriminate between cancer patients and healthy controls with a 100 % specificity and sensitivity. In addition, we also identified a potential predictor signature, which could be used to identify two cohorts of patients based on the overall survival. These novel findings demonstrated the tremendous potential of antibody microarrays in cancer research and clinical applications.

For more information please contact: Christer Wingren.

Johan Fransson: Functionally-associated target antigens in cancer. Defined by human antibodies and small interfering RNAs

(2006)

Cancer is the collective name for diseases that have one thing in common ? uncontrolled cell growth. The increase in cancer cell proliferation eventually results in tumor formation and, in the worst case, metastatic spread to other organs. Common treatment modalities of cancer include chemotherapy and radiation therapy. However, these methods are associated with severe side effects as well as induced drug resistance. Accordingly, to improve cancer prognoses, the introduction of innovative therapies, based on novel cancer targets, is needed.

This thesis is based upon four original papers, in which we address this issue and seek for new targets for cancer intervention. Specifically, in Paper I, we used phage display to isolate a set of antibodies that bound to breast and pancreatic carcinoma cells, and upon binding to the surface receptors, induced internalization into the cells. In Paper II, we employed the technology described in Paper I to isolate an internalizing antibody, denoted INCA-X, toward pancreatic carcinoma cells. The corresponding antigen was identified as the Ku70/80 heterodimer, a nuclear protein that could also be localized on the cell surface of tumor cells, where it has previously been suggested to function in adhesion, migration and invasion processes. The anti-Ku70/80 antibody (INCA-X) was shown to be a good delivery vehicle for cytotoxic drugs into several different cancer cell lines (pancreatic, breast, prostate, colorectal cancer). In Paper III, we identified three receptors that upon antibody crosslinking could induce apoptosis in B cell lymphoma cell lines. These antigens were HLA-DR/DP, IgM, and ICAM-1. Finally, in paper IV, we used DNA microarray technology to generate a list of 46 genes that are overexpressed in mantle cell lymphoma compared to normal B cells. From this list, three target genes were identified, based on loss-of-function screening after silencing of the corresponding transcripts by siRNA. In conclusion, by employing differential proteomic and genomic approaches, several functionally-associated targets were identified. By interfering with these antigens (via antibodies or siRNAs), changes in cancer cell physiology were induced, thus pointing toward a potential for tumor intervention.

For more information please contact: Carl Borrebaeck.

Cornelia Steinhauer: Protein microarrays based on single framework recombinant antibody fragments (SINFABS) - catcher and carrier - a crucial combination

(2005)

Antibody-based microarrays are among the novel class of rapidly evolving proteomic technologies. In recent years, antibody microarrays have emerged as a unique tool for high-throughput protein expression profiling with great promise within biomedicine and a wide range of potential applications, including disease diagnostics and biomarker discovery. In order to evolve the technology from small dedicated microarrays to high-density well-performing arrays for true global proteome analysis, this thesis focussed on the design of the two key components of the antibody microarray setup: the probe and the solid support, i.e. ?catcher and carrier?. The thesis is based upon five original papers that deal with i) the central features of the probe (on-chip functionality and stability, sensitivity and immobilisation strategies) and ii) the biocompatibility of the solid support (defined by the spot morphology, binding capacity, signal to noise ratio, dynamic range, sensitivity, reproducibility and the long-term performance). Single-framework recombinant antibody fragments (sinFabs), selected from the nCoDeR-library, were found to fulfil most, if not all, of the prerequisites required by well-performing probes for high-density antibody microarrays. In more detail, sinFabs were shown to be functional and stable on the chip with a constant on-chip performance (paper I and II) and to provide sensitive detection of low-abundant analytes (paper III). Furthermore, they could be genetically engineered for improved affinity coupling, thereby enabling non-purified probes to be coupled in an orientated way and purified directly on the chip (paper III and IV). Although sinFab microarrays were shown to be biocompatible with several commercially available and in-house designed solid supports of different structure (1-, 2- and 3-dimensional) and coupling chemistry (adsorption, covalent coupling and affinity binding), no single surface was found to fulfil all the requirements for a sensitive and high-density antibody microarray analysis (paper II and V). Thus, the development of a novel highly biocompatible macroporous silicon support coated with nitrocellulose (MAP3-NC7) (paper V) was a first step towards the specific design of optimised antibody microarray supports.

For more information please contact: Christer Wingren.

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Carl-Magnus Högerkorp: Genomic and molecular aspects of mature human B cells

(2005)

This thesis covers aspects of the mature human B cell and its different guises on its way to become a fully differentiated antibody forming plasma cell (AFC), the key player in the humoral immune system. The understanding of the complex molecular mechanisms taking place in the B cell, as it first encounters antigen, enters the germinal center (GC) reaction, giving rise to memory cells and plasma cells, is not only of importance for the curious scientist, but will also benefit the understanding of B cells in disease. For instance, what is the cause of B cell lymphomas and what role does B cells play in the etiology of autoimmunity? These questions and many more will be greatly facilitated by gaining knowledge of the molecular events influencing healthy B cells. This thesis is based on four original papers (I-IV) which deals with several different aspects of the mature human B cell. In paper I we looked into the properties of CD44 signaling and its role in B cell activation. Naive B cells isolated from human tonsils and sorted by FACS, were cultured in vitro for 3 days, stimulated with anti-BCR, and anti-CD40 stimuli in condition one and with an additional anti-CD44 stimuli in condition two. B cells, sampled at 6, 24 and 72 hours from each condition were analyzed by high-density oligonucleotide microarray analysis. The results demonstrated a role for CD44 in immunomodulation and inflammation, since transcripts for molecules such as IL-6 and IL-1alpha were specifically up-regulated by CD44. In paper II, by using high-density oligonucleotide microarrays, we analyzed mantel cell lymphomas (MCL) in relation to normal mature B cell subsets comprising naive, pre-activated, germinal center and memory cell subsets. From this analysis we could conclude that the malignant transformation event of MCLs appears to be an event occurring during the transition from a primary B cell follicle to a germinal center. In paper III we looked closer into what differs between human GC centroblasts and centrocytes isolated by FACS. By using high-density oligonucleotide microarray analysis and flow cytometry we concluded that the current phenotypic description of the different GC B cell subsets is misleading and that the centrocyte population represents a heterogeneous subset of cells comprising centroblasts, centrocytes and plasmablasts. In paper IV we extended our analysis of the mature tonsillar B cell subsets from paper II and III to also include plasmacells, GC founder cells, sub-epithelial cells and GC B cells described by other means. The microarray analysis grouped cells into three main categories: proliferating, non-proliferating and plasma cells. Cell type specific transcription programs were characterized.

For more information please contact: Carl Borrebaeck.

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Malin Lindstedt: Human dendritic cells and the regulation of allergic immune responses

(2005)

Dendritic cells (DC) are a heterogeneous lineage of bone-marrow-derived leukocytes that serve as the link between innate and adaptive immunity. They are professional antigen-presenting cells that play an important protective and regulatory role in both health and disease. This thesis is based upon six original papers that deal with the function and transcription of human DCs, with focus on their role in the inflammatory immune responses, such as allergic rhinitis. Transcriptional profiling of DCs with microarrays has been an extensively utilized technique in the projects presented in this thesis. We have evaluated the gene expression profiles and functions of two in vitro models of human DCs, namely the monocyte-derived DCs and the differentiated cell line MUTZ-3, and the transcriptional regulation induced in these models by pro-inflammatory signals. We have also studied the effect of allergenic stimulation on the transcription and function of DCs derived from healthy and allergic individuals. Two allergens were used, the detergent enzyme lipase and grass pollen, to evaluate the direct effect on phenotype and gene expression of DCs in addition to the subsequent ability of allergen-challenged DCs to amplify and modify the autologous effector T cell response. We demonstrate that the transcriptional responses of DCs and effector T cells to allergenic stimulation are different between allergic and healthy individuals. These transcriptional profiles involved in the immune recognition of allergens will be further evaluated in order to understand the interplay between DCs and T cells in allergic rhinitis. Furthermore, in addition to the in vitro models studied, we have performed phenotypical and transcriptional characterizations of in vivo DCs isolated from peripheral blood and tonsillar tissue. We suggest that follicular DCs in tonsils may have previously unacknowledged costimulatory functions in the germinal center reaction, as they express CD137. An extensive transcriptional profiling of freshly sorted DC subsets from blood and tonsils identified DC-subset selective gene expression and pinpoint their relationships. We demonstrate innate specialization of these subsets and show that the environment in tonsils determines the transcriptional activity of myeloid DCs. In conclusion, these studies have provided insight in the transcription and phenotype of in vivo immature/mature DC populations as well as in the immune response induced by allergens or inflammatory signals.


For more information please contact: Malin Lindstedt.

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Fredrik Karlsson: The biology of filamentous phage infection ­ implications for display technology

(2004)

Phage display technology is a biotechnological tool that can be used to obtain molecules/reagents able to discriminate between target molecules. Reagents with such features can be used in diverse settings like biological chemistry, diagnostics and in therapeutic applications. Phage display technology is based on the use of recombinant DNA technology, bacteria and a special kind of bacterial virus, the filamentous phage. However, the chance of obtaining high binding strength (affinity) reagents using conventional phage display is limited by e. g. the selection procedure. Therefore, protocols that increase the likelihood of recovering high affinity binding proteins from a pool of similar molecules, a so called library, are of interest. One approach is to modify the selection procedure of phage display, which has generated a special application called selective infection. Another approach is to study the infection process of filamentous phage, in hope of revealing mechanistic events suitable to manipulation. In this thesis, which is based on four original papers, selective infection is evaluated and the infection mechanism of filamentous phage is assessed in conjunction to implications for phage display technology.
By analysing the prerequisite for successful selective infection, we found a correlation between the affinity of the interacting pairs and infection efficiency and that a phage format allowing multiple display of binding proteins was superior to one that does not allow such multiple display. Thus, selective infection has characteristics that make it suitable for retrieving mainly high affinity binding proteins from phage display libraries. The infection mechanism of filamentous phage was dissected by analysing the molecular interactions between phage coat protein pIII and the bacterial co-receptor protein of phage infection, TolA. The binding affinities between these proteins and their different domains were characterised and novel interactions were detected, allowing for a refined hypothetical model of the infection mechanism of filamentous phage. Furthermore, TolA mutants were created for the analysis of its phage receptor and outer membrane integrity functions. It was found that these two TolA functions could be segregated, thus emphasising the mechanistic differences between the two functions of TolA. Finally, the changes in gene expression of phage infected Escherichia coli, was monitored by global transcription analysis and it was demonstrated that several host genes were co-ordinately affected. In conclusion, these studies have provided a basis for the development of phage display technology, as well as insights into several different aspects of the infection process of filamentous phage.


For more information please contact: Carl Borrebaeck.

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Sara Ek: Transcriptional analysis of mantle cell lymphoma - a global search for cellular origin and therapeutic targets

(2004)

This thesis is based upon four original papers describing the characterisation of mantle cell lymphoma (MCL) tumours and cell lines in comparison with different populations of normal B cells. Gene expression profiling, which were used in the studies, has during recent years developed as an important tool in characterisation of different types of malignancies. In this thesis, the technique is used to identify gene signatures in addition to providing information about the mRNA expression of more than 10 000 genes in the samples analysed. We found indications of an antigen-activated origin of MCL, in contrast to the current dogma. De-regulation of apoptosis and proliferation-associated pathways were further identified and possibly therapeutic targets were recognised. In an attempt to define the differences between high and low proliferative MCL, a proliferation-associated signature was established. The genes or gene-products constituting the signature are potential targets for therapy, as the genes are associated with proliferation, which is related to decreased survival time. Furthermore, MCL cell lines were studied to determine their usefulness as in vitro models for MCL. Hierarchical clustering, using a MCL-specific gene list, confirmed that the cell lines had preserved the expression of MCL-associated genes, as they were readily separate from other lymphoma cell lines, primary follicular lymphoma samples and normal B cells. However, one of the MCL-derived cell lines showed less correlation to the primary MCL tumours than the other cell lines. The same cell line carried a mutated VH gene, in contrast to the other MCL cell lines, and may be more suitable as in vitro model for MCL with mutated than unmutated VH genes.

For more information please contact: Sara Ek.

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Peter Ellmark: The CD40 receptor - target, tool and technology

(2002)

CD40 is a cell surface receptor of pivotal importance that is expressed on several of the cells in the immune system. It is critical for many important events, such as T cell dependent B cell activation, isotype switching, somatic mutation and generation of B cell memory. The central role of CD40 in the immune system makes it an ideal target for antibody based immunotherapy. This led us to characterise a panel of monoclonal anti-CD40 antibodies. In PAPER I, we investigated their cellular activation potential and analysed to what extent this correlates with their affinity, epitope specificity and domain recognition profile. The antibody profiles we obtained in this first study may be valuable for understanding of the mechanisms that influence the therapeutic capacity of these antibodies. In fact, one of the antibodies that we investigated is currently in phase I/II trials. However, all of the antibodies that we characterised in PAPER I are of mouse origin, which probably limits their clinical efficiency, due to the human anti-mouse response that most patients develop against such antibodies. Therefore we selected a set of human anti-CD40 antibodies, which are described in PAPER II, from a recombinant antibody gene library. These antibodies display a wide variety of distinct properties, which may make them a valuable source when evaluating therapeutic candidates for in vivo trials.

In PAPER III, we have used some of the anti-CD40 antibodies described in PAPER II to create an antibody library that was utilised to investigate antibody evolution in vitro. The results from this study showed that events, which resembles receptor revision, i.e. secondary rearrangements of antibody genes in the periphery, may provide an evolving antibody with competitive advantages during a selection process that is similar to the affinity maturation process in vivo. Our data reinforce the suggestion that receptor revision is an important complement to point mutations and insertions and deletions in the somatic hypermutation process that occur in germinal centres.

It has been suggested that members of the TNFR family pre-associate in the membrane via one of the extracellular domains, the pre-ligand assembly domain (PLAD). Therefore, in PAPER IV, we investigated the functional role of the different domains of CD40, in a B cell model system. The results from this study showed that neither of the extracellular domains is essential for signal transduction and, furthermore, implies that conformational changes play no critical role for the CD40 signalling pathway. Based on the findings that all of the extracellular part of CD40 can be replaced with retained signalling capacity, we developed a novel selection method, named Selection of Protein Interactions by Receptor Engagement (SPIRE). In PAPER V, we demonstrated that this selection system can be used for clonal enrichment of cells that display a mock-CD40 receptor, used as prey, on the surface by interaction with a certain bait protein. Thus, SPIRE allows for clonal selection of interacting protein pairs in a mammalian environment. SPIRE may have several different applications such as identification of tumour antigens or for molecular evolution of complex proteins.

For more information please contact: Carl Borrebaeck.

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Johan Lantto: Antibody evolution and repertoire development

(2002)

Antibodies are key players of the immune system in higher vertebrates, which provide a defense against potentially lethal threats from the environment. Besides their importance in the immune defense, antibodies have a great potential as reagents in biological chemistry and diagnostics, and as therapeutic agents against both infectious diseases and cancer. Despite tremendous advances in all fields regarding antibodies, the understanding of the processes that shape antibody responses in vivo is far from complete. In this thesis, which is based on five original papers, I present work that deals with aspects of the evolution of antibodies and the development of antibody repertoires.

By analyzing the inherited repertoires of human genes encoding the variable domains of antibodies, I have discovered regions with accumulations of repetitive trinucleotide motifs. These repeats were mainly found in the complementarity determining regions (CDR), and most likely target them with insertions and deletions during the maturation of an antibody response. In order to investigate the functional consequences of such modifications, insertions and deletions were introduced into the CDR of antibody fragments by molecular engineering. The results showed that modifications of this kind are well tolerated in the CDR of the heavy chain, and can be utilized to expand sequence and structure space of an antigen-binding site beyond what is encoded by the germline gene repertoire. Thus, insertions and deletions seem to be an efficient way of expanding antibody sequence and structure space both in vivo and in vitro, and it is conceivable that these modifications can also be used in antibody engineering to create antibodies against specific targets.

I have also studied the development of antibodies reactive with a weakly immunogenic epitope on human cytomegalovirus, and what the critical parameters are that determine the neutralizing capacity of such antibodies. The results from these studies showed that the genes encoding one of the two existing human antibodies reactive with this epitope do not have the intrinsic features required for high-affinity interaction with the epitope, as mutations are required at key positions. The poor immunogenicity of this epitope may in fact be a consequence of the lack of an imprinted specificity in the human germline repertoire. Furthermore, the obtained data demonstrated that a divalent antibody format is required for effective virus neutralization via this epitope, and that the neutralizing potential of a repertoire of antibodies reactive with this epitope is determined by the reaction rate kinetics and fine-specificity of the interaction with the epitope. These results provide insights into the development of human antibody repertoires against weakly immunogenic targets, and are also discussed in the context of vaccine development.

For more information please contact: Mats Ohlin.

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Bengt Johansson-Lindbom: Regulatory properties of dendritic cells and B cells in adaptive immunity

(2002)

This thesis is based upon four original papers in which human dendritic cells (DCs) and B cells have been analyzed in terms of how they influence the character of adaptive immune responses. DCs isolated from human tonsils were found to possess a capacity to directly regulate proliferation, isotype switching, and antibody production in B cells. DC-produced cytokines, including IL-13, were identified as critical mediators of these B cell responses. Furthermore, gene chip technology was used to evaluate the nature and kinetics of the global gene expression taking place in monocyte-derived DCs exposed to inflammatory agents. Obtained results revealed an extensive and temporal reprogramming of these cells in response to TNF-a, IL-1b, plus mediators released by activated monocytes. The altered gene expression was represented by a pronounced upregulation of a number of mRNAs encoding proteins with established functions in the regulation of both T and B cell responses. This transcriptional reorganization may reflect the effect of in vivo released inflammatory mediators, indicating that DCs can be fully matured to activate adaptive immunity in response to tissue inflammation. Furthermore, also the role of B cells in immune regulation was investigated. Antigen-activated B cells within germinal centers (GC) were found to produce the Th2-polarizing cytokine IL-4 and consequently they could elicit Th2-differentiation in vitro. In agreement with this in vitro observation, a Th2 precursor subset was identified in human tonsil and demonstrated to uniformly display a GC-associated CXCR5high phenotype. Therefore Th2-development in human tonsils appears to selectively occur within GCs and to be supported by B cells secreting IL-4. Moreover, IL-4-producing B cells were also identified within follicles located in colon mucosa, indicating that B cell-dependent Th2 development can take place in several of the mucosa associated lymphoid tissues. Finally, functional properties of the previously described CD57+ GC Th cells were addressed and obtained results showed that these cells represent anergized T cells. These data thus suggest that B cells and GCs regulate CD4+ T cell differentiation in a finely tuned fashion, either by promoting differentiation of Th2 cells or by furnishing T cell-unresponsiveness. In conclusion, I propose that Th cell polarization may be subjected to a counterbalanced regulation, where DC-produced IL-12 and/or IFN-a/b promote Th1-differentiation, whereas GCs and B cells preferentially furnish Th2-development but also contribute to suppression of T cell responses.

For more information please contact: Carl Borrebaeck.

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Pernilla Jirholt: Generation and evolution of human antibody fragments - The CDR shuffling approach

(2000)

Antibodies are important proteins participating in a number of reactions to protect us from harmful diseases. Apart from this important function, they are also useful in different technical and medical applications. This, however, relies on our possibility to produce antibodies in vitro. In the first part of my thesis a new technology called CDR shuffling was developed to create diverse and highly functional antibody libraries. Specific antibody fragments were isolated from such CDR shuffling antibody libraries by phage display selections.
When isolating antibody fragments from antibody libraries, the retrieved clones may not have the desired reactivity characteristics. It may be necessary to further in vitro evolve the clones to achieve the desired kinetic or specificity parameters. In the second part of my thesis, the CDR shuffling technology was used to further evolve initially isolated antibody fragments. By this method, I was able to define a central core structure important for generating a mucin-1 specificity. By keeping this central core and introducing variability in other parts of the molecules, I further improved the kinetic parameters of the isolated clones.
In conclusion, this thesis presents a novel technology suitable for the generation and evolution of human antibody fragments.

For more information please contact: Mats Ohlin

Magnus Strandh: Insights into weak affinity antibody-antigen interactions. Studies using affinity chromatography and optical biosensors.

(2000)

Molecular recognition is fundamental for the function of biological systems. The properties of the participating biomolecules dictate the type of forces, the strength and the dynamics involved in the interaction. Many interactions are very strong whereas others exhibit weak affinity. Typically, weak interactions work in concert to trigger a biological response. The advantage with this approach is the inherent dynamics. It has been shown that this approach can be successful for in vitro applications as well. By exposing analytes to a multitude of specific, weak affinity interactions, which are governed by fast association and dissociation rates, separation based on small differences in affinity is possible. The same principles can also be used to characterize biological weak affinity binders and for analytical purposes. This thesis has discussed biomolecular interactions in the weak affinity range (defined in this investigation as dissociation constants (KD) larger than 10 mM) in general and has focused on how they can be studied and exploited in vitro. Weak affinity monoclonal antibodies were used as model systems in three different applications: (i) to explore how weak affinity chromatography based on monoclonal IgM can be employed to separate structurally related steroids under non-denaturing conditions. (ii) to investigate the possibilities of studying weak interactions between antibodies and haptens with an optical biosensor based on surface plasmon resonance. A rationale for the design of such experiments to avoid inaccurate results was suggested. (iii) to introduce continuous real-time immunosensing for monitoring fluctuating concentrations of biomolecules in a flow.

For more information please contact: Carl Borrebaeck

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Nina Nilsson: In vivo and in vitro evolution of molecular mechanisms - Importance in B cell development and phage display

(2000)

Bone marrow resident hematopoietic stem cells differentiate into all different types of lymphoid populations, there among mature naive B cells expressing IgM and IgD. During the maturation, the B cells are subjected to several steps of selection to ensure proper development of the antigen receptor. The mechanisms regulating the selection are to great extent unknown. In paper I of this thesis, we demonstrate the presence of the Fas system in late stages of B lymphopoiesis and further discuss the implication of these findings.

In paper II, we investigated the molecular events accompanying the apoptotic process observed in pre-B cells following cross-linking of MHC class I. We were able to show that the apoptotic process was not linked to activation of protein kinase C nor to changes in cytosolic Ca2+ concentrations, intracellular events common in cells undergoing apoptosis. We utilised the differential display technology to detect two genes important in MHC class I mediated apoptosis.

The objective in paper III was to functionally dissect the protein responsible for the infection of bacterial cells by filamentous bacteriophage i.e. the adsorption protein. The improved understanding of the mechanisms behind the infection process could be utilised in the construction of phage display systems used for improved protein expression, as in paper IV, or for studying protein - protein interactions, as in paper V.

For more information please contact: Carl Borrebaeck

Eva Andersson: Studies of T- and B-cells for the generation of human antigen specific antibodies

(1998)

Human antibodies generated in vitro may be highly valuable for diagnostic and therapeutic purposes. For a long time most human antibodies produced in vitro have been of the IgM isotype with rather low affinities. In this thesis a method for production of IgG switched antigen-specific human antibodies is presented. This is a two step protocol, in which the first step is a selection procedure of donors with sufficient T-cell reactivity against tetanus toxoid (TT). The culture consists of a primary and a secondary immunization, of peripheral blood cells, where the secondary step includes CD40 activation together with antigen and preactivated T cells. The antigen is a heterotope with one B- and one T-cell (TT) epitope. In the model system 60 % of the donors produced anti-V3 specific IgG antibodies. The immunization system was used to study the humoral response against the tumor related antigen Mucin 1. Cells from the immunization were used for preparation of an Ab gene library, which was screened by using phage display. The selected Fab fragments were of low affinity with high dissociation rate constants. Mucin 1 gave a lower response, in this immunization system, compared to V3 probably due to the repetitive nature of the antigen. The in vitro immunization system somewhat mimics the germinal center reaction in vivo. In the germinal center B cells mutate their antibody genes, are selected by their ability to bind, take up and process antigen, and differentiate into plasma cells or memory cells. The germinal center reaction is dependent on T cells and a fraction of the CD4+ T cells in the germinal center coexpresses CD57. We have studied this population and a population in peripheral blood which also coexpresses CD4 and CD57. The GC derived population was able to stimulate B cells to Ig production when IL-2 was added while the blood derived population could not. The GC population was also able to promote survival of GC B cells, in vitro, to the same extent as CD57- Th cells. Furthermore the GC population expressed the costimulatory molecule, CD28 while the blood population did not. Neither GC nor blood derived CD57+ cells expressed any detectable amounts of mRNA specific for IL-2, IL-4 or IFN-g.

For more information please contact: Carl Borrebaeck

Katarina Dahlenborg: Cellular and molecular aspects of the germinal centre reaction

(1998)

The humoral immune response to a T cell dependent protein antigen will over time generate antibodies with an increased affinity to the antigen. This is due to a process known as affinity maturation and proceeds in a, for this purpose, well designed micro-milieu within secondary lymphoid organs known as germinal centres. The increase in affinity of an antibody in, e.g. mouse and man is due mainly to incorporation of single base-pair mutations, so called somatic mutations, in combination with an efficient competitive re-selection of better binders on the antigen held in its naÔve form on follicular dendritic cells.
Studies on affinity-maturation in human B cells and in cells within the germinal centre, have been hampered by the fact that the cells of interest die rapidly in vitro and the lack of a proper cell culture system for study of such cells. The aim of this thesis is to design such a cell culture system that allows growth of single human germinal centre B cells, known to accumulate somatic mutations in vivo, to study these single cell cultures for in vitro mutational activity, and to investigate signals required to induce naÔve B cells towards a germinal centre B cell phenotype in vitro. By the use of a rapid screening system for in vitro accumulation of somatic mutations (RT-PCR and SSCP), clones raised from single cells were analysed for in vitro mutational activity.
Human tonsillar, in vivo primed, germinal centre B cells were cultured together with murine EL-4 cells and shown to preserve some of their most characteristic extracellular phenotype and proliferation capacity. Still, no further in vitro accumulation of mutations were found in such single cell culture clones. Though, combination with interleukin-supplemented cultures based on CD40 crosslinking (via CD32 transfected murine L cells) of human GC-B cells revealed that their mutational activity could be preserved with one of the interleukin-combinations used, pointing out the importance of soluble factors for preserving the germinal centre reaction as well. In addition, signals required in vitro to differentiate resting B cells to a germinal centre B cell phenotype were identified. It was shown that stimulation via sIg and CD40, in combination with CD44, seem to drive the B-cell towards a GC-B cell phenotype. The role for CD4+CD57+ GC-T cells were also examined, and were shown to be able to rescue GC-B cells in vitro and drive peripheral blood B cells to Ig production by the addition of exogenous IL-2.

For more information please contact: Carl Borrebaeck

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Sigurdur Ingvarsson: Signalling mechanisms in B cell differentiation. Studies on specific human immune response in vitro

(1998)

We need to understand the major signalling events in human B cell differentiation in order to be able to regulate the generation of human antigen specific antibodies.
This study has focused on the signalling mechanisms involved in generation of germinal centre reaction and secretion of specific antibodies by human B cells, using different in vitro immunisation technologies. Naive human B cells that were stimulated by crosslinking their sIg, CD40 and CD44 acquired a germinal centre phenotype. Signalling via these surface molecules caused increased proliferation and made the B cells enter an apoptotic cycle, which is characteristic for germinal centre B cells. Three different in vitro immunisation protocols were utilised to study the mechanisms behind secretion of human antigen specific antibodies. First, soluble antigen in the cultures was shown to abrogate specific Ig response by reducing the number of memory B cells secreting specific antibodies to a recall antigen. By crosslinking the antigen bound to the sIg of those B cells the antigen specific suppression could be lifted. Secondly, purified B cells together with isolated CD4 T cells and a superantigen could be induced to secrete antibodies to primary antigens, by crosslinking the antigen bound to their sIg. The specific antibody secretion was totally dependent on CD40 and CD86 ligation of the B cells. The third protocol demonstrates specific IgE production using a system that induces isotype switching in vitro. Specific IgE production appears to be dependent on NFkappaB activation by IL-4, produced and secreted by Th2 cells generated during the primary response.
This thesis provides possible signalling mechanisms for the initiation of germinal centre reactions. It also presents data on antibody production in vitro in terms of antigen presentation and cell-cell interaction.

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Ann-Christin Malmborg: Molecular recognition in antibody engineering. Studies on recombinant and phage displayed antibodies

(1996)

In vitro generation of antibody fragments of desired specificity and affinity plays an important role since it permits the generation of reagents which may be valuable for diagnostic and therapeutic applications. Furthermore, the kinetic parameters for an antibody-antigen interaction, rather than the affinity, has shown to correlate with biological functions like virus neutralization. This points at the importance of being able to evaluate kinetic parameters. In this thesis we have evaluated the BIAcore™ biosensor for measuring affinity and kinetic constants and pointed at events where precautions need to be taken. The recently launched ORIGEN Analyser was, furthermore, evaluated for measuring affinity constants and a protocol for measuring dissociation rate constants was developed. The ability to select antibodies from phage displayed antibody libraries based on their kinetic parameters would be valuable. Two approaches in this direction were taken. The first was to use BIAcore™ biosensor for selection. By collecting elution fractions of an injected phage displayed antibody library, it could be shown that the time of dissociation is proportional to the dissociation rate constant. The second approach was to develop a modified protocol for SAP selection. SAP (selection and amplification of phage) links specific interaction to phage infection, by displaying the antibody fragment on a non-infectious phage and the antigen, fused to protein3, is added free in solution. Thus, only specific phages are allowed to infect and are amplified. We showed that addition of competing antigen to the interaction between antigen-protein3 fusion protein and a model phage displayed antibody library favoured low dissociation rate constants. Furthermore, a reduction in interaction time favoured high association rate constants. Thus, it was, for the first time, possible to select antibody fragments based on association rate. The last part ofthe thesis presents a new concept, which integrates phage and bacterial display in anattempt to physically link the genetic information of specifically interacting antibody-antigen molecules. This was performed by expressing peptide antigens on the surface of E. coli F pilus. The peptides were expressed as a fusion to pilin, the building block of pilus and encoded by the traA gene, which completely blocked protein3-mediated wild type phage infection. However, when a phage displayed scFv antibody was allowed to interact with pilus displayed peptide, we obtained a bacterial infection mediated by the specific antibody-antigen interaction. Thus, specific interaction on a protein-ligand level could be genetically rescued as a cellular linkage of the involved genes.This principle could have the potential to allow for screening of specific molecular interactions by crossing one phage and one bacterial library.

For more information please contact: Carl Borrebaeck

Marie Wallén-Öhman: MHC class I-induced apoptosis in hematopoietic cells

(1996)

The aims of this study have been focused on the identification of cell surface structures involved in induction and regulation of apoptosis. Monoclonal antibodies against the major histocompatibility complex class I (MHC-I) were shown to induce apoptosis when cross-linked on the cell surface of different malignant cells having a pre-B or a myeloid precursor phenotype. The apoptotic response was not epitope dependent, since several different anti-MHC-I antibodies, reacting with different monomorphic determinants of the a chain or b2-microglobulin all induced apoptosis in these cells. However, external cross-linking of antibodies was strictly required for the apoptotic effect. Among cells originating from mature peripheral blood lymphocytes, anti-CD40 stimulated B-cells were susceptible to anti-MHC-I induced apoptosis, whereas resting as well as superantigen activated B- and T- cells were non-responsive to MHC-I ligation. The apoptotic process was not linked to protein kinase C activation or changes in cytoplasmic calcium concentration. In situ terminal doxynucleotidyl transferase staining of apoptotic cells at various stages during MHC-I induced cell death revealed that apoptosis occurred predominantly in the G2/M phase of the cell cycle, with the first apoptotic cells appearing after approximately 12 hours of incubation. The results suggest a role for MHC-I mediated apoptosis during differentiation and activation of certain hematopoietic cells.

For more information please contact: Carl Borrebaeck

Christina Furebring: Expression of Ig genes. Regulation of transcription and production of human antibodies

(1996)

During B lymphocyte development, the transcriptional activity of the IgH locus is subject to spatial and temporal changes. The 3' enhancer (3'E) has been suggested to play an important role in regulation of immunoglobulin gene expression late in B cell development. We have investigated, using transgenic mice, the role of the 3'E in regulating Ig gene expression. Mice harbouring a rearranged IgH gene potentiated by the VH promoter in combination with the IgH intron enhancer (µE), the 3'E or the µE/3'E pair were generated. The 3'E activity is mainly observed in lymphoid tissues and is mainly restricted to the in vivo activated B cells. The 3'E can potentiate Ig gene expression directly in conjunction with the VH promoter. The expression level of the µE/3'E controlled transgene is fivefold higher as compared to the transgene controlled by the µE alone.
The aim of my subsequent studies has been to generate and produce human monoclonal antibodies. We have focused our interest on immortalization of the variable region genes, from hybridoma or from a single antigen-specific B cell, using the powerful PCR technique. The variable region genes from single B cells can be immortalized directly or after a cellular amplification step, involving the EL-4 and CD40 cell culture systems. The variable region genes obtained can thereafter be expressed either as Ab fragments, in prokaryotic host cells, or as the entire Ab, in eukaryotic host cells. To allow efficient expression of intact Ab we have optimized a eukaryotic IgH gene expression vector using different combinations of regulatory elements.

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Ann Catrin Simonsson Lagerkvist: In vitro generation and selection of antigen-specific antibodies

(1996)

The present investigation has focused on in vitro generation and selection of antigen-specific human antibodies and the introduction of additional V region diversity by mutagenesis. In paper I the effect of exogenous lymphokines and a lysosomotropic agent on Ig secretion and lymphokine mRNA expression was investigated. In accordance with earlier results it was found that LeuLeuOMe treatment of PBL removed certain cell populations that had a negative influence on the in vitro response, in this case the lymphokine mRNA expression pattern and kinetics. Paper II and III describe two different culture systems for specific responses, either with immobilized antibodies to CD3 as polyclonal activator, or with SEA. We have shown that crosslinking sIg with antigen is required for specific responses to occur in these culture systems. With SEA-mediated culture both primary and secondary response antigen give rise to specific antibodies. Paper IV-V describe selection of specific antibodies either directly from PBL using antigen-coated magnetic beads or as phage-displayed antibodies. GroELS chaperonins increase the copynumber of phages in the phagestocks, facilitating subsequent isolation of rare clones. Finally, in paper VI, a method for introducing diversity into preexisting antibodies using nucleotide doped PCR primers is presented and we show that antibody affinity is improved. This thesis describes diversification of the technologies for producing human monoclonal and recombinant antibodies. This results in an extended choice in approaching the problem of acquiring a certain specificity and then engineering this to taste, with respect to isotype and affinity. Cellular and molecular biological approaches have both been used. These complement each other rather that constituting an either/or solution.

For more information please contact: Carl Borrebaeck

Peter Ifversen: Human antibody technology. An evaluation of the SCID-hu-PBL model

(1995)

Transplantation of human lymphoid cells into mice suffering from severe combined immunodeficiency (SCID mice) results in spontaneous production of human antibodies. We have investigated the ability of SCID-hu-PBL mice to induce antigen-specific primary and secondary human immune responses against various antigens, for the purpose of generating human monoclonal antibodies of desired specificities. SCID mice were transplanted with peripheral blood lymphocytes from tetanus toxoid (TT) immune donors, thus recruiting specific T cell help from TT specific T cells by physically coupling the B cell antigen with TT. In order to demonstrate that T cell help were retrieved from antigen-specific T cells, a TT specific T helper cell clone was established. Only mice transplanted with splenocytes and the autologous T cell clone were able elicit human antigen-specific responses. Improvement of T cell help was obtained by in vitro priming human B cells with a superantigen (SEA) before transplantation with autologous T cells. Human IgM and IgG responses were obtained against the hapten dinitrophenyl (DNP), ovine submaxillary mucin (OSM), the melanoma-asscociated gangliosides GM2 and GD2 and a V3 loop peptide from gp120 (HIV-1). Gangliosides were introduced to SCID-hu-PBL as a liposome construct with TT encapsulated into the liposomes and very high human IgG serotiters were obtained against the normally weak immunogenic GD2 ganglioside. Further, terminal differentiation of antigen-specific human B cells was indicated, as well as problems of retrieving the generated specific B cells, which limits the use of the model with respect to generate human monoclonal antibodies. However, the ability of the SCID-hu-PBL model to elicit human immune responses emphasize the use of SCID-hu-PBL as a vaccine model. Furthermore the results suggest a potential value of the liposome construct as a melanoma vaccine in clinical trials of melanoma patients. In order to obtain longterm culture of antibody producing B cell lines, Epstein-Barr virus is a valuable tool to immortalize the generated B cell specificity. However, EBV transformed B cells are often unstable in longterm cultures and difficult to clone. We proved the ability of the B cell growthfactor thioredoxin, secreted by the T cell hybridoma MP6, to increase the cloning efficacy EBV infected B cells.

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Christina Eriksson: The SCID mouse. A tool for generation of human monoclonal antibodies?

(1995)

Since Köhler and Milstein first developed methods for obtaining mouse antibodies, a number of technologies have been developed to overcome problems related to the production of high-affinity human monoclonal antibodies directed against selected antigens. The aim of this work was to evaluate the SCID mouse, allowing in vivo growth of human tissue, as a tool for generation of human antibodies of desired specificity. SCID mice were transplanted with human peipheral blood lymphocytes, immunized with either a recall antigen or primary antigens, whereafter the human immune response was evaluated as the presence of human Ig or antigen specific antibodies in sera of the mice. Two techniques, in order to generate primary antibody responses, were developed. In the first, primary antigens were physically linked to the recall antigen tetanus toxoid (TT), in order to recruit sufficient T cell help for specific antibody production. The second approach involved priming of human B cells with a superantigen, SEA, prior to transplantation into SCID mice, in combination with autologous T cells. SEA is a very potent activator of T cells when presented on MHC class II expressing accessory cells.

Depletion of human NK cells from human PBL, by treatment with LeuLeuOMe, prior to transplantation into SCID mice resulted in increased titers of human Ig and the Ig repertoire resembled that of the donor as measured by kappa/lambda ratios of human light chains. As T helper cells, expressing CD45R0, are known to support B cell proliferation and differentiation, production of human Ig in SCID mice were shown to require cotransplantation of CD45R0+ T cells and B cells. The SCID mice produced specific antibodies in a dose-dependent manner when immunizing with TT. Specific antibodies of high avidity were obtained when immunizing with a low dose of antigen, whereas a higher dose resulted in production of antibodies of lower avidity. However, immunizing with even higher doses of antigen, resulted in a total abrogation of the specifc immune response. Primary immune responses could be obtained against the hapten dinitrophenyl (DNP), the V3 loop peptide derived from glycoprotein (gp120) (HIV-1), the melanoma-assocoated GD2 ganglioside and ovine submaxillary mucin (OSM), when using the two approaches described above. Human immune B cells, recovered from immunized SCID mice, could be transformed with EBV and yielded lymphoblasoid cell lines producing antigen specific human IgG antibodies. Thus, the SCID mouse provides a promising tool for the generation of human monoclonal antibodies of desired specificity.

For more information please contact: Carl Borrebaeck

Marta Dueñas: Phage display and bacterial expression of antibody fragments

(1995)

As the development of monoclonal antibody technologies, the generation of genetic engineered antibodies has created an explosion of knowledge of the antibody molecule and has provided a powerful tool for the study of immunological responses. The present investigation has been focused on the development of new technologies for the generation, selection and characterization of antibody fragments in Escherichia coli. Plasmid vectors were designed for intra and extracellular expression of different antibody fragments. Increased level of intracellular expression was achieved after over-expression of Hsp 60/10 but not specific activity was found in the cytoplasmic fraction. In one case, a single nucleotide mutation in an antibody gene strongly influenced the expression levels, studies at the mRNA and protein levels . Over-expression of the chaperonins was evaluated on bacteriophage displaying antibody fragments on the surface and it was found favor the phage assembly by increasing one hundred times the phage titer. A new method for selection of antibody fragments displayed on filamentous bacteriophages was designed based on linking antigenic recognition and phage replication by means of a mutant helper phage and a soluble fusion protein. For its evaluation in this new system of selection, mutant helper phages derived from M13 bacteriophage were constructed and evaluated. By using the SAP system we constructed, selected and characterized antibody libraries from in vitro immunized B cells and studied features of this novel immune response. It was, furthermore, shown that by manipulating antigen binding conditions and by introducing an antigen chase different procedures were established that allowed a selection based on kinetic parameters (affinity as well as on and off rates) of the displayed antibody fragments.

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Li-te Chin: Site-directed in vitro immunization. A model of sequential antigen-specific activation of human B cells

(1994)

Culture conditions and signal requirements for primary and secondary antigen-specific B-cell antibody responses of human peripheral blood mononuclear cells to the HIV-1 principal neutralization domain (PND) were delineated. It was found that the optimal immunogen for primary in vitro immunization to generate antigen-specific IgM encompasses heterologous epitopes (heterotope) that consists of both a promiscous T-cell epitope derived from tetanus toxin (ptt) and a B-cell epitope derived from the PND (pV3). We therefor characterized the T-helper functions by the production of T-cell clones specific to tetanus toxoidl. It was found that cloned T cells with TH0-like lymphokine pattern may play an important regulatory role in the antigen-specific humoral immune response both in vivo and in vitro. In an attempt to recruit the T helper functions to an in vitro immunization system, cloned T helper cells specific to ptt were thus established. Induction of antigen-specific IgG responses by antigen re-exposure in vitro required, however, the simultaneous presence of autologous ptt-specific CD4+ T cells and an additional signal to B cells via CD40. A sequential induction in vitro model was established by mimicking the response of lymphoid organs to antigen. The production of antibodies with switched isotype (IgG) and high affinity has thus been developed. The resultant antibodies not only showed accumulated genetic variation in association with affinity maturation, but also projected the binding relationship between the V3 loop and its possible receptor, i. e. CD26 in some cases. Synthetic peptides corresponding to CDR regions of the neutralizing antibody showed specific binding to the synthetic V3 loop as well as neutralization of viral infection in vitro. Application of this site-directed in vitro immunization technology might prove critical in the design of immunotherapeutics for human infectious diseases.

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Karin Kristensson: T cell mediated effects on in vitro B cell responses

(1993)

The generation of an in vitro immune response requires specific interactions between T and B cells. In this study CD4+ T-helper cells were further divided into subsets according to their different reactivity with antibodies specific to the CD45RA and CD45R0 molecules of the leucocyte common antigen (LCA). The two different subsets of T-helper cells have been characterized concerning their ability to induce B cell proliferation and B celldifferentiation into Ig secreting cells. The production of various cytokines, such as IL-2, IL-4, IL-5 and IFN-g, in response to polyclonal activators such as Staphylococcal Enterotoxin A (SEA) have also been studied. The T cells subsets were sorted out from peripheral blood mononuclear cells by use of a fluorescence activated cell sorter (FACS) and their respective effector functions assayed together with B cells. We have been able to show that CD45R0 cells represent the true T-helper cell which is able to support B cell proliferation as well as differentiation. In respect of b cell proliferation, the difference between the two T cell populations were shown to be dose dependent, in that CD45RA+ T cells required higher concentrations of SEA in order to support this. The ability to support B cell proliferation were shown to correlate with the expression of CD40 Ligand.
The CD45R0+ T cells were shown to produce a number of cytokines (IL-2, IL-4, IL-5 and IFN-g) after primary stimulation, whereas the CD45RA+ T cells produced mainly IL-2. When stimulated in vitro the CD45RA+ T cells gained expression of the CD45R0 molecule with a concomitant loss of CD45RA. These in vitro differentiated cells also gained functional properties of the CD45R0+ cells, i.e. ability to support B cell proliferation and differentiation. After differentiation, the former CD45RA+ T cells also produced cytokines characteristic of the CD45R0 subset. The difference in ability to support B cell responses has also been confirmed in an in vivo system using mice that suffer from severe combined immuno deficiency (SCID). Production of human Ig i these mice were shown to require cotransplantation of CD45R0+ T cells and B cells.

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Mats Ohlin: Human Monoclonal Antibody Technology. A tool to investigate human antibody repertoires

(1992)

The development of technologies to generate cell lines producing monoclonal antibodies of human origin has a great impact on the ability to study repertoires of specificities in the humoral immune response. Furthermore, it permits the generation of reagents which may be valuable for diagnostic and therapeutic applications and which lack some of the disadvantages of similar murine antibodies. In this study Epstein-Barr virus (EBV)-transformation of peripheral blood mononuclear cells (PBMC) has been used to immortalize human B lymphocytes. A novel technique to eliminate the capacity of PBMC to inhibit the outgrowth of EBV-transformed B cells is described. This technique, which employs pretreatment of PBMC with L-leucyl-L-leucine methyl ester, removes the inhibitory effect mediated by T cell populations in vitro. Furthermore, it is shown that the treatment impairs the ability of CD4+ T lymphocytes to respond to EBV-infected B cells by production of mRNA encoding the outgrowth-inhibitory lymphokine interferon-gamma. This technique has been used to allow establishment of lymphoblastoid cell lines (LCL) producing antigen-specific human antibodies from PBMC obtained from non-immunized donors, following in vitro stimulation. The LCL were subsequently fused to an immortal heteromyeloma fusion partner in order to rescue the antibody specificities of the potentially unstable EBV-transformed cell lines. Monoclonal antibodies recognizing digoxin or recombinant fragments of HIV-1 related glycoproteins gp41/gp120 have thus been obtained and characterized with respect to fine specificity using epitope mapping. These antibodies are of the low-affinity IgM type although the genes encoding the immunoglobulin variable regions show evidence of mutations in relation to previously described germ line sequences. Despite the low affinity of these immunoglobulins, some of the HIV-1 gp120-specific antibodies are able to block spreading of virus between cells in vitro thus suggesting an ability of antibodies established by these approaches from naive/primary repertoires to mediate biologically important functions. Human monoclonal antibody-producing cell lines secreting immunoglobulins recognizing either phosphoprotein (pp65) or glycoprotein (gB) of cytomegalovirus (CMV) have similarly been estabished from lymphocytes derived from seropositive donors. The antigens recognized by these antibodies have previously been suggested to be important for the diagnosis of CMV infection and for the humoral immune surveillance of CMV-related infections, respectively. Conformationsl as well as apparently linear epitopes which are detected by human antibody repertoires have been characterized. In addition, the neutralizing capacity of antibodies recognizing different fine-specificities expressed by glycoprotein B is described.

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Lena Danielsson: Human Monoclonal Antibodies. In vitro Immunization and Cloning of DNA Encoding Variable Regions

(1990)

Production of human monoclonal antibodies has long been hampered by problems to obtain properly immunized cells and stable antibody-producing hybridomas. In vitro immunization technology would prove useful as a mean to obtain lymphocytes immunized against therapeutically useful antigens. An in vitro immunization procedure which allows human peripheral blood B lymphocytes to be activated was therefor developed. The crucial step was the removal of lysosome-rich cells in the peripheral blood cell compartment, such as large granular lymphocytes, monocytes/macrophages, cytotoxic T cells and a subset of CD8+ cells. This was obtained by treating the cells with L-leucine methyl ester or its lysosomal condensation product L-leucyl-L-leucine methyl ester. The cells were then cultivated for 6-7 days in the presence of antigen. The culture was supported by a supernatant from pokeweed mitogen-stimulated and irradiated T cells, and by interleukin 2. The immunized cellswere then immortalized. Different fusion partners were used; a mouse myeloma, a human lymphoblastoid cell line, or a huma-mouse heteromyeloma. Of those, the heteromyeloma proved to be superior. The positive effect of the lysosomotropic agents on the outgrowth of Epstein-Barr virus infected B cells was also evaluated.
In order to obtain a basis for structure-function studies, protein engineering and expression of immunoglobulins, a polymerase chain reaction (PCR)-based method allowing rapid isolation of DNA encoding immunoglobulin variable regions was developed. This approach allowed cloning and sequence determination of DNA fragments for both mu and gamma heavy chains, and kappa and lambda light chains. Optimization of the RNA isolation procedure and the PCR method made it possible to obtin DNA encoding the variable regions from a single hybridoma cell.

For more information please contact: Carl Borrebaeck

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