| 1. |
- Bondza, Sina, et al.
(författare)
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Bivalent binding on cells varies between anti-CD20 antibodies and is dose-dependent
- 2020
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Ingår i: mAbs. - : TAYLOR & FRANCIS INC. - 1942-0862 .- 1942-0870. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Based on their mechanism of action, two types of anti-CD20 antibodies are distinguished: Type I, which efficiently mediate complement-dependent cytotoxicity, and Type II, which instead are more efficient in inducing direct cell death. Several molecular characteristics of these antibodies have been suggested to underlie these different biological functions, one of these being the manner of binding to CD20 expressed on malignant B cells. However, the exact binding model on cells is unclear. In this study, the binding mechanism of the Type I therapeutic antibodies rituximab (RTX) and ofatumumab (OFA) and the Type II antibody obinutuzumab (OBI) were established by real-time interaction analysis on live cells. It was found that the degree of bivalent stabilization differed for the antibodies: OFA was stabilized the most, followed by RTX and then OBI, which had the least amount of bivalent stabilization. Bivalency inversely correlated with binding dynamics for the antibodies, with OBI displaying the most dynamic binding pattern, followed by RTX and OFA. For RTX and OBI, bivalency and binding dynamics were concentration dependent; at higher concentrations the interactions were more dynamic, whereas the percentage of antibodies that bound bivalent was less, resulting in concentration-dependent apparent affinities. This was barely noticeable for OFA, as almost all molecules bound bivalently at the tested concentrations. We conclude that the degree of bivalent binding positively correlates with the complement recruiting capacity of the investigated CD20 antibodies.
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| 2. |
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| 3. |
- Bondza, Sina, et al.
(författare)
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Complement-Dependent Activity of CD20-Specific IgG Correlates With Bivalent Antigen Binding and C1q Binding Strength
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies directed against the CD20 surface antigen on B cells are widely used in the therapy of B cell malignancies. Upon administration, the antibodies bind to CD20 expressing B cells and induce their depletion via cell- and complement-dependent cytotoxicity or by induction of direct cell killing. The three antibodies currently most often used in the clinic are Rituximab (RTX), Ofatumumab (OFA) and Obinutuzumab (OBI). Even though these antibodies are all of the human IgG1 subclass, they have previously been described to vary considerably in the effector functions involved in therapeutic B cell depletion, especially in regards to complement activation. Whereas OFA is known to strongly induce complement-dependent cytotoxicity, OBI is described to be far less efficient. In contrast, the role of complement in RTX-induced B cell depletion is still under debate. Some of this dissent might come from the use of different in vitro systems for characterization of antibody effector functions. We therefore set out to systematically compare antibody as well as C1q binding and complement-activation by RTX, OFA and OBI on human B cell lines that differ in expression levels of CD20 and complement-regulatory proteins as well as human primary B cells. Applying real-time interaction analysis, we show that the overall strength of C1q binding to live target cells coated with antibodies positively correlated with the degree of bivalent binding for the antibodies to CD20. Kinetic analysis revealed that C1q exhibits two binding modes with distinct affinities and binding stabilities, with exact numbers varying both between antibodies and cell lines. Furthermore, complement-dependent cell killing by RTX and OBI was highly cell-line dependent, whereas the superior complement-dependent cytotoxicity by OFA was independent of the target B cells. All three antibodies were able to initiate deposition of C3b on the B cell surface, although to varying extent. This suggests that complement activation occurs but might not necessarily lead to induction of complement-dependent cytotoxicity. This activation could, however, initiate complement-dependent phagocytosis as an alternative mechanism of therapeutic B cell depletion.
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| 4. |
- Bondza, Sina, et al.
(författare)
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Conjugation Effects on Antibody-Drug Conjugates : Evaluation of Interaction Kinetics in Real Time on Living Cells
- 2014
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 11:11, s. 4154-4163
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-drug conjugates (ADC) have shown promising effects in cancer therapy by combining the target specificity of an antibody with the toxicity of a chemotherapeutic drug. As the number of therapeutic antibodies is significantly larger than those used as ADCs, there is unused potential for more effective therapies. However, the conjugation of an additional molecule to an antibody may affect the interaction with its target, altering association rate, dissociation rate, or both. Any changes of the binding kinetics can have subsequent effects on the efficacy of the ADCs, thus the kinetics are important to monitor during ADC development and production. This paper describes a method for the analysis of conjugation effects on antibody binding to its antigen, using the instrument LigandTracer and a fluorescent monovalent anti-IgG binder denoted FIBA, which did not affect the interaction. All measurements were done in real time using living cells which naturally expressed the antigens. With this method the binding profiles of different conjugations of the therapeutic anti-EGFR antibody cetuximab and the anti-CD44v6 antibody fragment AbD15171 were evaluated and compared. Even comparatively small modifications of cetuximab altered the interaction with the epidermal growth factor receptor (EGFR). In contrast, no impact on the AbD15171-CD44v6 interaction was observed upon conjugation. This illustrates the importance to study the binding profile for each ADC combination, as it is difficult to draw any general conclusion about conjugation effects. The modification of interaction kinetics through conjugation opens up new possibilities when optimizing an antibody or an ADC, since the conjugations can be used to create a binding profile more apt for a specific clinical need.
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| 5. |
- Bondza, Sina
(författare)
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Deciphering Binding Patterns of Therapeutic Antibodies with Immune Cells : From Method Development to Application
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Reversible binding, for example between signaling molecules and receptors on the cell surface, is one of the main means to communicate information in cellular systems. Knowledge about how molecules interact is crucial for both understanding biological function and for therapeutic intervention. The cellular environment often makes ligand-receptor interactions complex with the membrane providing structural support and containing other components that interfere with the interaction. One of the fastest growing drug classes for targeting cellular receptors are monoclonal antibodies (mAb), in particular within oncology. Therapeutic mAbs can have direct effects on target cells mediated via the Fab-domain and immune-related effects that are mediated via the Fc-domain. An example of the latter is activation of the complement system by binding of its first component C1q to Fc-domains. Furthermore, immune cells can recognize Fc-domains via Fc-receptors and cause target cell death by a process called antibody-dependent cellular cytotoxicity (ADCC).Increased understanding about structure-binding-function relationships facilitates rational drug design, as has been demonstrated with the development of next-generation mAbs that harbor a structural modification on their Fc-domain that strengthens the interaction with immune cells thereby increasing ADCC efficacy. In this thesis, assays for characterizing mAb binding and mAb mediated interactions on live cells were developed and applied to illustrate how detailed knowledge about binding processes helps to understand the relation between binding and biological function.Paper I describes a protocol for real-time interaction analysis of antibodies with live immune cells enabling binding measurements in a relevant cellular context with the data resolution needed to study complex binding processes.Paper II presents a novel real-time proximity assay that allows to study binding kinetics in connection with receptor dimerization and clustering thereby aiding in decipher complex interactions.In paper III, binding patterns of the CD20 mAbs rituximab, ofatumumab and obinituzumab were established on cells revealing that the fraction of bivalently bound mAbs differed resulting in dose-dependent affinities for rituximab and obinituzumab.In paper IV, a C1q binding assay to mAb opsonized cells was developed and it was shown that a higher degree of bivalent binding correlated with stronger C1q binding for the CD20 mAbs evaluated in paper III.In paper V, an assay to study mAb mediated cell-cell interactions was set-up and it was found that neutrophil engagement with target cells was similar for antibodies of IgG and IgA isotype.
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| 6. |
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| 7. |
- Bondza, Sina, et al.
(författare)
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Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells
- 2017
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:24, s. 13212-13218
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Tidskriftsartikel (refereegranskat)abstract
- Cellular receptor activity is often controlled through complex mechanisms involving interactions with multiple molecules, which can be soluble ligands and/or other cell surface molecules. In this study, we combine a fluorescence-based technology for real-time interaction analysis with fluorescence quenching to create a novel time-resolved proximity assay to study protein-receptor interactions on living cells. This assay extracts the binding kinetics and affinity for two proteins if they bind in proximity on the cell surface. One application of real-time proximity interaction analysis is to study relative levels of receptor dimerization. The method was primarily evaluated using the HER2 binding antibodies Trastuzumab and Pertuzumab and two EGFR binding antibodies including Cetuximab. Using Cetuximab and Trastuzumab, proximity of EGFR and HER2 was investigated before and after treatment of cells with the tyrosine-kinase inhibitor Gefitinib. Treated cells displayed 50% increased proximity signal, whereas the binding characteristics of the two antibodies were not significantly affected, implying an increase in the EGFR-HER2 dimer level. These results demonstrate that real-time proximity interaction analysis enables determination of the interaction rate constants and affinity of two ligands while simultaneously quantifying their relative colocalization on living cells.
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| 8. |
- Bondza, Sina, et al.
(författare)
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Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells
- 2017
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Understanding molecular interactions on immune cells is crucial for drug development to treat cancer and autoimmune diseases. When characterizing molecular interactions, the use of a relevant living model system is important, as processes such as receptor oligomerization and clustering can influence binding patterns. We developed a protocol to enable time-resolved analysis of ligand binding to receptors on living suspension cells. Different suspension cell lines and weakly adhering cells were tethered to Petri dishes with the help of a biomolecular anchor molecule, and antibody binding was analyzed using LigandTracer. The protocol and assay described in this report were used to characterize interactions involving eight cell lines. Experiments were successfully conducted in three different laboratories, demonstrating the robustness of the protocol. For various antibodies, affinities and kinetic rate constants were obtained for binding to CD20 on both Daudi and Ramos B-cells, the T-cell co-receptor CD3 on Jurkat cells, and the Fc gamma receptor CD32 on transfected HEK293 cells, respectively. Analyzing the binding of Rituximab to B-cells resulted in an affinity of 0.7-0.9 nM, which is similar to values reported previously for living B-cells. However, we observed a heterogeneous behavior for Rituximab interacting with B-cells, which to our knowledge has not been described previously. The understanding of complex interactions will be facilitated with the possibility to characterize binding processes in real-time on living immune cells. This provides the chance to broaden the understanding of how binding kinetics relate to biological function.
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| 9. |
- Brandsma, Arianne M., et al.
(författare)
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Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG
- 2019
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Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Antibody therapy of cancer is increasingly used in the clinic and has improved patient's life expectancy. Except for immune checkpoint inhibition, the mode of action of many antibodies is to recognize overexpressed or specific tumor antigens and initiate either direct F(ab')(2)-mediated tumor cell killing, or Fc-mediated effects such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/P) after binding to activating Fc receptors. All antibodies used in the clinic are of the IgG isotype. The IgA isotype can, however, also elicit powerful anti-tumor responses through engagement of the activating Fc receptor for monomeric IgA (Fc alpha RI). In addition to monocytes, macrophages and eosinophils as Fc alpha RI expressing immune cells, neutrophils are especially vigorous in eliminating IgA opsonized tumor cells. However, with IgG as single agent it appears almost impossible to activate neutrophils efficiently, as we have visualized by live cell imaging of tumor cell killing. In this study, we investigated Fc receptor expression, binding and signaling to clarify why triggering of neutrophils by IgA is more efficient than by IgG. Fc alpha RI expression on neutrophils is similar to 2 times and similar to 20 times lower than that of Fc gamma receptors Fc gamma RIIa and Fc gamma RIIIb, but still, binding of neutrophils to IgA- or IgG-coated surfaces was similar. In addition, our data suggest that IgA- mediated binding of neutrophils is more stable compared to IgG. IgA engagement of neutrophils elicited stronger Fc receptor signaling than IgG as indicated by measuring the p-ERK signaling molecule. We propose that the higher stoichiometry of IgA to the Fc alpha R/FcR gamma-chain complex, activating four ITAMs (Immunoreceptor Tyrosine-based Activating Motifs) compared to a single ITAM for Fc gamma RIIa, combined with a possible decoy role of the highly expressed Fc gamma RIIIb, explains why IgA is much better than IgG at triggering tumor cell killing by neutrophils. We anticipate that harnessing the vast population of neutrophils by the use of IgA monoclonal antibodies can be a valuable addition to the growing arsenal of antibody-based therapeutics for cancer treatment.
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| 10. |
- Mansouri, Larry, et al.
(författare)
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Functional loss of I kappa B epsilon leads to NF-kappa B deregulation in aggressive chronic lymphocytic leukemia
- 2015
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 212:6, s. 833-843
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Tidskriftsartikel (refereegranskat)abstract
- NF-kappa B is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-kappa B pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes I kappa B epsilon, a negative regulator of NF-kappa B in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced I kappa B epsilon protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that I kappa B epsilon loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-kappa B deregulation during lymphomagenesis.
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| 11. |
- Mansouri, Larry, et al.
(författare)
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Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia.
- 2015
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 212:6, s. 833-843
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Tidskriftsartikel (refereegranskat)abstract
- NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.
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| 12. |
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| 13. |
- Petersen, Inga, et al.
(författare)
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Multivalent design of the monoclonal SynO2 antibody improves binding strength to soluble α-Synuclein aggregates
- 2023
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Ingår i: mAbs. - : Taylor & Francis. - 1942-0862 .- 1942-0870. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Soluble aggregates are reported to be the most neurotoxic species of alpha-Synuclein (alpha Syn) in Parkinson's disease (PD) and hence are a promising target for diagnosis and treatment of PD. However, the predominantly intracellular location of alpha Syn limits its accessibility, especially for antibody-based molecules and prompts the need for exceptionally strong soluble alpha Syn aggregate binders to enhance their sensitivity and efficacy for targeting the extracellular alpha Syn pool. In this study, we have created the multivalent antibodies TetraSynO2 and HexaSynO2, derived from the alpha Syn oligomer-specific antibody SynO2, to increase avidity binding to soluble alpha Syn aggregate species through more binding sites in close proximity. The multivalency was achieved through recombinant fusion of single-chain variable fragments of SynO2 to the antibodies' original N-termini. Our ELISA results indicated a 20-fold increased binding strength of the multivalent formats to alpha Syn aggregates, while binding to alpha Syn monomers and unspecific binding to amyloid beta protofibrils remained low. Kinetic analysis using LigandTracer revealed that only 80% of SynO2 bound bivalently to soluble aSyn aggregates, whereas the proportion of TetraSynO2 and HexaSynO2 binding bi- or multivalently to soluble alpha Syn aggregates was increased to similar to 95% and 100%, respectively. The overall improved binding strength of TetraSynO2 and HexaSynO2 implies great potential for immunotherapeutic and diagnostic applications with targets of limited accessibility, like extra-cellular alpha Syn aggregates. The ability of the multivalent antibodies to bind a wider range of alpha Syn aggregate species, which are not targetable by conventional bivalent antibodies, thus could allow for an earlier and more effective intervention in the progression of PD.
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| 14. |
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| 15. |
- Weber, Judith, et al.
(författare)
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Modelling ligand depletion for simultaneous affinity and binding site quantification on cells and tissue
- 2023
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- The quantification of the number of targets in biological systems is an important parameter to assess the suitability of surface markers as targets for drugs, drug delivery and medical imaging. Likewise, quantifying the interaction with the target in terms of affinity and binding kinetics is essential during drug development. Commonly used approaches to quantify membrane antigens on live cells are based on manual saturation techniques that are labour-intensive, require careful calibration of the generated signal and do not quantify the binding rates. Here, we present how measuring interactions in real-time on live cells and tissue under ligand depletion conditions can be used to simultaneously quantify the kinetic binding parameters as well as the number of available binding sites in a biological system. Suitable assay design was explored with simulated data and feasibility of the method verified with experimental data for exemplary low molecular weight peptide and antibody radiotracers as well as fluorescent antibodies. In addition to revealing the number of accessible target sites and improving the accuracy of binding kinetics and affinities, the presented method does not require knowledge about the absolute signal generated per ligand molecule. This enables a simplified workflow for use with both radioligands and fluorescent binders.
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