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- Carlsson, Fredrika, et al.
(författare)
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A dimerized single-chain variable fragment system for the assessment of neutralizing activity of phage display-selected antibody fragments specific for cytomegalovirus.
- 2012
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 376:Online 01 Dec 2011, s. 69-78
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Tidskriftsartikel (refereegranskat)abstract
- Abstract in UndeterminedCytomegalovirus (CMV) causes severe sequelae in congenitally infected newborns and may cause life-threatening disease in immuno-deficient patients. Recent findings demonstrate the possibility to alleviate the disease by infusing intravenous immunoglobulin G (IgG) preparations, indicating that antibodies are an effective therapeutic option. Modern molecular methodologies, like phage display, allow for the development of specific antibodies targeting virtually any antigen, including those of CMV. However, such methodologies do not in general result in products that by themselves mediate biological activity. To facilitate a semi-high-throughput approach for functional screening in future efforts to develop efficacious antibodies against CMV, we have integrated two different approaches to circumvent potential bottlenecks in such efforts. Firstly, we explored an approach that permits easy transfer of antibody fragment encoding genes from commonly used phage display vectors into vectors for the production of divalent immunoglobulins. Secondly, we demonstrate that such proteins can be applied in a novel reporter-based neutralization assay to establish a proof-of-concept workflow for the generation of neutralizing antibodies against CMV. We validated our approach by showing that divalent antibodies raised against the antigenic domain (AD)-2 region of gB effectively neutralized three different CMV strains (AD169, Towne and TB40/E), whereas two antibodies against the AD-1 region of gB displayed minor neutralizing capabilities. In conclusion, the methods investigated in this proof-of-concept study enables for a semi-high-throughput workflow in the screening and investigation of biological active antibodies.
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| 2. |
- Carlsson, Fredrika
(författare)
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Antibody Development - Examples from viral and proteomic studies
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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 enrichment of proteins by antibody fragments 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.
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| 3. |
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| 4. |
- Carlsson, Fredrika, et al.
(författare)
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Humoral immunity targeting site I of antigenic domain 2 of glycoprotein B upon immunization with different cytomegalovirus candidate vaccines.
- 2007
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Ingår i: Vaccine. - : Elsevier BV. - 1873-2518 .- 0264-410X. ; 26:1, s. 41-46
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Tidskriftsartikel (refereegranskat)abstract
- Glycoprotein B (gB) is a major component in several vaccines that are under development for prevention of disease by cytomegalovirus. It contains multiple determinants that are targets for neutralizing antibodies. One of them is site I of antigenic domain 2 (AD-2). The epitope, defined by short peptides, is quite conserved between different isolates. However, it is poorly immunogenic in natural infection. In this study we investigated the extent to which different vaccines, attenuated live Towne vaccine with or without priming with a canarypox virus coding for gB, or a recombinant gB vaccine adjuvanted with MF59, induced antibodies to this epitope. As in natural infection only a fraction of all subjects developed antibody responses against site I of AD-2 following vaccination. We suggest that strategies that enhance immunogenicity of this epitope will improve vaccine efficacy.
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| 6. |
- Carlsson, Fredrika, et al.
(författare)
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Novel antibody specificities targeting glycoprotein B of cytomegalovirus identified by molecular library technology
- 2009
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 25:6, s. 429-436
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies provide some protection against cytomegalovirus-mediated disease. All aspects of antibody recognition of important viral antigens are however not fully appreciated. Glycoprotein B (gB), a key protein in the viral membrane, participates in viral infection and it is a component of prototype vaccines. By using combinatorial antibody library and selection technology, novel antibody specificities targeting gB have now been isolated. We define a monoclonal antibody fragment able to recognize site I of antigenic domain (AD) 2, a poorly immunogenic epitope targeted by potent virus-neutralizing antibodies, in a way that is different from the binding of antibodies induced by infection but similar to those induced by vaccination. We also describe the existence of a novel epitope overlapping site I of AD-2 and AD-1, the immunodominant epitope of gB. These specificities, derived by molecular engineering, will be useful for the future assessment of humoral immune responses against this opportunistic viral infection.
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| 9. |
- Hägerbrand, Karin, et al.
(författare)
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Bispecific antibodies targeting CD40 and tumor-associated antigens promote cross-priming of T cells resulting in an antitumor response superior to monospecific antibodies
- 2022
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Ingår i: Journal for ImmunoTherapy of Cancer. - : BMJ. - 2051-1426. ; 10:11
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Tidskriftsartikel (refereegranskat)abstract
- Background Indications with poor T-cell infiltration or deficiencies in T-cell priming and associated unresponsiveness to established immunotherapies represent an unmet medical need in oncology. CD40-targeting therapies designed to enhance antigen presentation, generate new tumor-specific T cells, and activate tumor-infiltrating myeloid cells to remodel the tumor microenvironment, represent a promising opportunity to meet this need. In this study, we present the first in vivo data supporting a role for tumor-associated antigen (TAA)-mediated uptake and cross-presentation of tumor antigens to enhance tumor-specific T-cell priming using CD40×TAA bispecific antibodies, a concept we named Neo-X-Prime. Methods Bispecific antibodies targeting CD40 and either of two cell-surface expressed TAA, carcinoembryonic antigen-related cell adhesion molecule 5 (CEA) or epithelial cell adhesion molecule (EpCAM), were developed in a tetravalent format. TAA-conditional CD40 agonism, activation of tumor-infiltrating immune cells, antitumor efficacy and the role of delivery of tumor-derived material such as extracellular vesicles, tumor debris and exosomes by the CD40×TAA bispecific antibodies were demonstrated in vitro using primary human and murine cells and in vivo using human CD40 transgenic mice with different tumor models. Results The results showed that the CD40×TAA bispecific antibodies induced TAA-conditional CD40 activation both in vitro and in vivo. Further, it was demonstrated in vitro that they induced clustering of tumor debris and CD40-expressing cells in a dose-dependent manner and superior T-cell priming when added to dendritic cells (DC), ovalbumin (OVA)-specific T cells and OVA-containing tumor debris or exosomes. The antitumor activity of the Neo-X-Prime bispecific antibodies was demonstrated to be significantly superior to the monospecific CD40 antibody, and the resulting T-cell dependent antitumor immunity was directed to tumor antigens other than the TAA used for targeting (EpCAM). Conclusions The data presented herein support the hypothesis that CD40×TAA bispecific antibodies can engage tumor-derived vesicles containing tumor neoantigens to myeloid cells such as DCs resulting in an improved DC-mediated cross-priming of tumor-specific CD8 + T cells. Thus, this principle may offer therapeutics strategies to enhance tumor-specific T-cell immunity and associated clinical benefit in indications characterized by poor T-cell infiltration or deficiencies in T-cell priming.
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