| 1. |
- Liljenfeldt, Lina, 1983-
(author)
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CD40L Gene Therapy for Solid Tumors
- 2014
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Doctoral thesis (other academic/artistic)abstract
- Adenoviral CD40L gene therapy (AdCD40L) is a strong inducer of anti-tumor immune responses via its activation of dendritic cells (DCs). Activated DCs can in turn activate T cells, which are key players in an efficient anti-tumor response.This thesis includes three papers that focus on different aspects of AdCD40L gene therapy. In the first paper, the infiltration of suppressive CD11b+Gr-1+ cells in orthotopic MB49 bladder tumors was investigated and found to be significantly reduced while activated T cells were increased when the tumors had been treated with local AdCD40L gene therapy. Further, AdCD40L could tilt the cells in the tumor microenvironment in favor of an efficient anti-tumor immunity (M1 macrophages and activated T cells) instead of an immunosuppressive environment (CD11b+Gr-1int/low myeloid cells and M2 macrophages).Immunotherapy combined with chemotherapy has shown promising results, and the second paper investigates the combination of AdCD40L gene therapy together with the chemotherapeutic drug 5-Fluorouracil (5-FU). A synergistic effect of the combination treatment on orthotopic MB49 bladder tumors could be demonstrated. The combination therapy resulted in decreased tumor growth, increased survival and systemic MB49-specific immunity, whereas AdCD40L or 5-FU therapy alone had a poor effect on tumor growth.Efficient AdCD40L therapy is dependent on high transduction efficiency in both cancer cells and cells present in the tumor microenvironment. In an attempt to enhance the transduction efficiency, and thereby the therapeutic efficacy, a modified adenovirus was developed for paper three. This modified Ad5PTDf35(mCD40L) could, in comparison with the unmodified Ad5(mCD40L), demonstrate increased transduction capacity of a variety of murine cells. Further, the ability of antigen presenting cells (APCs) to present antigens to T cells was improved after transduction with Ad5PTDf35(mCD40L).
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| 2. |
- Sandin, Linda, 1979-
(author)
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Immunomodulatory Therapy of Solid Tumors : With a Focus on Monoclonal Antibodies
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Cancer, historically considered a genetic disease, is currently acknowledged to affect the whole body. Our immune system is one key player that can elicit a response against malignant cells but can also promote tumorigenesis. Tumors avoid immune recognition by creating a suppressive microenvironment and inducing tolerance. T-cells are regarded a major effector cell type in tumor immunotherapy. An important ”switch” needed for T-cell activation involves so-called costimulatory and coinhibitory receptors. In this thesis, experimental tumor models were used to investigate the potential of immunomodulatory antibodies to stimulate immune cells and subsequently eliminate tumors.First, systemic antibody blockade of two negative checkpoint regulators (CTLA-4 and PD-1) present on T-cells was evaluated in combination with local CpG therapy or standard BCG treatment. Indeed, this combinatorial therapy with CpG augmented anti-tumor effects with increased levels of tumor-directed T-cells and reduced tumor-infiltrating Tregs.Secondly, as these immunomodulatory antibodies elicit severe side effects in patients, a local low-dose delivery regimen was explored as an alternative to systemic bolus treatment. Our results demonstrated that an approximately seven times lower dose of aCTLA-4, compared to systemic delivery, could eradicate both primary and distant tumors.CD40-expressing APCs are another potential target in antibody-mediated cancer therapy. CD40-stimulated dendritic cells (DCs) have the capability to activate tumor-directed T-cells to kill tumor cells. We next sought to investigate agonistic CD40 antibody efficacy and in vivo biodistribution when delivered locally compared to the equivalent systemic dose. Anti-tumor effects were dependent on CD8+ T-cells, host CD40 expression and the presence of tumor antigen at the injection site. CD40 antibodies were cleared from the circulation and accumulated in lymphoid organs, where, upon repeated aCD40 dosing, target APC populations increased in numbers and upregulated their surface CD40 expression.Lastly, CD40 agonist antibodies were mixed with nanoparticles to enhance their stimulatory properties. B-cells demonstrated increased proliferative capacity and DCs became more activated when exposed to the cocktail. Further, this combination reduced serum levels of pro-inflammatory cytokines compared to plain antibodies. The results herein advocate further exploratory studies of the delivery of monoclonal antibodies at the tumor site in order to improve anti-tumor effects and reduce toxicity.
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| 3. |
- Backman, Max, et al.
(author)
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Infiltration of NK and plasma cells is associated with a distinct immune subset in non‐small cell lung cancer
- 2021
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In: Journal of Pathology. - : John Wiley & Sons. - 0022-3417 .- 1096-9896. ; 255:3, s. 243-256
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Journal article (peer-reviewed)abstract
- Immune cells of the tumor microenvironment are central but erratic targets for immunotherapy. The aim of this study was to characterize novel patterns of immune cell infiltration in non-small cell lung cancer (NSCLC) in relation to its molecular and clinicopathologic characteristics. Lymphocytes (CD3+, CD4+, CD8+, CD20+, FOXP3+, CD45RO+), macrophages (CD163+), plasma cells (CD138+), NK cells (NKp46+), PD1+, and PD-L1+ were annotated on a tissue microarray including 357 NSCLC cases. Somatic mutations were analyzed by targeted sequencing for 82 genes and a tumor mutational load score was estimated. Transcriptomic immune patterns were established in 197 patients based on RNA sequencing data. The immune cell infiltration was variable and showed only poor association with specific mutations. The previously defined immune phenotypic patterns, desert, inflamed, and immune excluded, comprised 30, 13, and 57% of cases, respectively. Notably, mRNA immune activation and high estimated tumor mutational load were unique only for the inflamed pattern. However, in the unsupervised cluster analysis, including all immune cell markers, these conceptual patterns were only weakly reproduced. Instead, four immune classes were identified: (1) high immune cell infiltration, (2) high immune cell infiltration with abundance of CD20+ B cells, (3) low immune cell infiltration, and (4) a phenotype with an imprint of plasma cells and NK cells. This latter class was linked to better survival despite exhibiting low expression of immune response-related genes (e.g. CXCL9, GZMB, INFG, CTLA4). This compartment-specific immune cell analysis in the context of the molecular and clinical background of NSCLC reveals two previously unrecognized immune classes. A refined immune classification, including traits of the humoral and innate immune response, is important to define the immunogenic potency of NSCLC in the era of immunotherapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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| 4. |
- Burman, Joachim, 1974-
(author)
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Curing Multiple Sclerosis : How to do it and how to prove it
- 2014
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Doctoral thesis (other academic/artistic)abstract
- Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for multiple sclerosis (MS) with now more than 600 documented cases in the medical literature. Long-term remission can be achieved with this therapy, but when is it justified to claim that a patient is cured from MS? In attempt to answer this question, the outcome of the Swedish patients is described, mechanisms behind the therapeutic effect are discussed and new tools for demonstration of absence of disease have been developed.In Swedish patients treated with HSCT for aggressive MS, disease free survival was 68 % at five years, and no patient progressed after three years of stable disease. Presence of gadolinium enhancing lesions prior to HSCT was associated with a favorable outcome (disease free survival 79 % vs 46 %, p=0.028). There was no mortality and no patient required intensive care.The immune system of twelve of these patients was investigated further. In most respects HSCT-treated patients were similar to healthy controls, demonstrating normalization. In the presence of a potential antigen, leukocytes from HSCT-treated patients ceased producing pro-inflammatory IL-17 and increased production of the inhibitory cytokine TGF-β1 suggesting restoration of tolerance.Cytokine levels and biomarkers of tissue damage were investigated in cerebrospinal fluid from a cohort of MS patients. The levels were related to clinical and imaging findings. A cytokine signature of patients with relapsing-remitting MS could be identified, characterized by increased levels of CCL22, CXCL10, sCD40L, CXCL1 and CCL5 as well as down-regulation of CCL2. Further, we could demonstrate that active inflammation in relapsing-remitting MS is a tissue damaging process, with increased levels of myelin basic protein and neurofilament light. Importantly, relapsing-remitting MS patients in remission displayed no tissue damage. In secondary progressive MS, moderate tissue damage was present without signs of active inflammation.From a clinical vantage point, it seems that we confidently can claim cure of relapsing-remitting MS patients after five years absence of disease activity. The new tools for evaluation of disease can strengthen this assertion and may enable earlier prediction of outcome.
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| 5. |
- Eltahir, Mohamed
(author)
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Antibody-based Cancer Immunotherapy : Personalization, response prediction and safety considerations
- 2021
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Doctoral thesis (other academic/artistic)abstract
- Antibody-based therapeutics have remarkably improved the field of immuno-oncology. Multiple monoclonal antibodies (mAbs) are approved for clinical use, and numerous antibodies are under clinical development. The scope of this thesis is to study the personalization of antibody-based immunotherapeutics and tools to predict their efficacy and safety.In paper I, we investigated a new method for predicting immune toxicity related to mAbs infusion, the whole blood loop assay (WBLA). The assay recapitulates the in vivo setting and harmonizes well with clinically validated cytokine release assays (CRAs) following agonistic mAbs infusion. We also demonstrated the robustness of the assay in studying complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC).Rituximab, the first approved mAb for an oncology indication, is known to induce CRS occasionally. In paper II, the WBLA was used to profile the chronic lymphocytic leukemia (CLL) patients’ specific responses to rituximab infusion. We demonstrated rituximab-induced CRS profile and complement activation in blood from CLL patients but not in blood from healthy donors. We also noted that NK cells were a significant source of the rituximab-induced cytokine release. Using Fc mutant versions of rituximab, the mode-of-action of rituximab in whole blood with respect to CDC and ADCC was elaborated.In paper III, we presented a novel flexible peptide cancer vaccine platform based on an anti-CD40 agonistic antibody. The platform consists of a bispecific antibody targeting CD40 and peptide-tagged antigens. The bispecific antibody retained the agonistic activity of anti-CD40 and was superior to parental anti-CD40 mAb in targeting antigen cross-presentation and stimulating both CD8+ and CD4+ T cell responses.In paper IV, we investigated the feasibility of proximity-extension assay (PEA) plasma proteomic analysis in predicting response to checkpoint inhibitors (CPIs) in non-small cell lung cancer patients. CPIs show great success in the clinic. However, not all patients benefit from CPIs. Using an immuno-oncology protein panel, we demonstrated that high plasma levels of T cell activation proteins were associated with better survival. We also identified an association between the pre-CPI plasma levels of CXCL9, CXCL10, IL-15, ADA and Casp8 and the response to CPI therapy. In conclusion, this thesis demonstrates the feasibility of using the WBLA to assess antibody infusion efficacy and safety, as well as PEA plasma proteomics to predict response to CPI therapy. Additionally, it presents a novel approach for personalized therapeutic cancer vaccine delivery.
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| 6. |
- Mangsbo, Sara, 1981-
(author)
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Immunological Checkpoint Blockade and TLR Stimulation for Improved Cancer Therapy
- 2009
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Doctoral thesis (other academic/artistic)abstract
- This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.
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| 7. |
- Rofo, Fadi
(author)
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Enhancing the therapeutic effect of biological drugs with protein engineering : Focusing on pre-clinical Alzheimer’s disease therapy
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Aggregation of the amyloid-β peptide (Aβ) is one of the main pathological hallmarks in Alzheimer’s disease (AD). The soluble Aβ aggregates (oligomers and protofibrils) have shown to be the most harmful species. Hence, targeting these aggregates can be of therapeutic potential.Protein therapy is one of the fastest growing fields in drug development with more than 100 FDA approved protein-drugs in the last decade. Despite that, protein-drugs (mainly antibodies) targeting Aβ displayed limited beneficial effects in AD clinical trials. This might be attributed to the presence of the blood-brain barrier (BBB) that hinders the entry of big molecules such as proteins into the brain. In paper I, we fused somatostatin peptide (SST) to the previously developed BBB transporter (scFv8D3). The new protein, SST-scFv8D3, exhibited a 120-times longer plasma half-life compared to SST, and reached the brain at high levels when intravenously administered. When tested in APPswe mouse model of AD, SST-scFv8D3 significantly enhanced neprilysin (NEP)-mediated degradation of hippocampal Aβ42 after only three injections. In paper II, treatment with SST-scFv8D3 displayed a wide-ranging effect on AD brain proteome. Mitochondrial and neuronal growth proteins were among the most altered protein-groups, where SST-scFv8D treatment shifted them towards wild-type levels.There is potential to increase the binding strength and selectivity of antibodies to small Aβ aggregates (oligomers), which are thought to be the most toxic Aβ species. In paper III, we developed a multivalent antibody format with additional binding sites having short distances between them. The new antibody format displayed a 40-fold reduction in the dissociation rate from Aβ protofibrils. Furthermore, the multivalent antibody could strongly bind small Aβ oligomers, which has been difficult to achieve with conventional IgG antibodies. In paper IV, we developed a bispecific version of the multivalent antibody capable of passing the BBB. A single intravenous injection of the new antibody format was enough to significantly clear soluble Aβ aggregates from the brain of tg-ArcSwe mice. In paper V, we developed recombinant proteins with NEP linked to an Fc-fragment to provide long half-life and to the above-mentioned BBB transporter. When applied at therapeutic doses, these proteins significantly degraded plasma Aβ, but displayed limited effects on brain Aβ concentration, probably due to their short retention times in the brain.In conclusion, we developed new protein-drugs with improved binding properties to Aβ, ability to cross the BBB, and therapeutic potential in pre-clinical mouse models of AD.
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