Exploring complement-dependent cytotoxicity by rituximab isotypes in 2D and 3D-cultured B-cell lymphoma

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Abstract

Background

The therapeutic IgG1 anti-CD20 antibody, rituximab (RTX), has greatly improved prognosis of many B-cell malignancies. Despite its success, resistance has been reported and detailed knowledge of RTX mechanisms are lacking. Complement-dependent cytotoxicity (CDC) is one important mode of action of RTX. The aim of this study was to systematically evaluate factors influencing complement-mediated tumor cell killing by RTX.

Methods

Different RTX isotypes, IgG1, IgG3, IgA1 and IgA2 were evaluated and administered on four human CD20 ⁺ B-cell lymphoma cell lines, displaying diverse expression of CD20 and complement-regulatory protein CD59. Complement activation was assessed on lymphoma cells grown in 2 and 3-dimensional (3D) culture systems by trypan blue exclusion. CDC in 3D spheroids was additionally analyzed by Annexin V and propidium iodide staining by flow cytometry, and confocal imaging. Anti-CD59 antibody was used to evaluate influence of CD59 in RTX-mediated CDC responses. Statistical differences were determined by one-way ANOVA and Tukey post hoc test.

Results

We found that 3 out of 4 lymphomas were sensitive to RTX-mediated CDC when cultured in 2D, while 2 out of 4 when grown in 3D. RTX-IgG3 had the greatest CDC potential, followed by clinical standard RTX-IgG1 and RTX-IgA2, whereas RTX-IgA1 displayed no complement activation. Although the pattern of different RTX isotypes to induce CDC were similar in the sensitive lymphomas, the degree of cell killing differed. A greater CDC activity was seen in lymphoma cells with a higher CD20/CD59 expression ratio. These lymphomas were also sensitive to RTX when grown in 3D spheroids, although the CDC activity was substantially reduced compared to 2D cultures. Analysis of RTX-treated spheroids demonstrated apoptosis and necrosis essentially in the outer cell-layers. Neutralization of CD59 overcame resistance to RTX-mediated CDC in 2D-cultured lymphoma cells, but not in spheroids.

Conclusions

The results demonstrate that CDC outcome in CD20 ⁺ B-cell lymphoma is synergistically influenced by choice of RTX isotype, antigen density, tumor structure, and degree of CD59 expression. Assessment of tumor signatures, such as CD20/CD59 ratio, can be advantageous to predict CDC efficiency of RTX in vivo and may help to develop rational mAbs to raise response rates in patients.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-022-09772-1.

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Most cited references 46

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Understanding the tumor immune microenvironment (TIME) for effective therapy

Weiping Zou, T. Kevin Howcroft, Elisa C Woodhouse … (2018)

The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient’s tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.

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IgG Subclasses and Allotypes: From Structure to Effector Functions

Gestur Vidarsson, Gillian Dekkers, Theo Rispens (2014)

Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcγR) and C1q. As a result, the different subclasses have different effector functions, both in terms of triggering FcγR-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces. However, FcRn is also expressed in myeloid cells, where it participates in both phagocytosis and antigen presentation together with classical FcγR and complement. How these properties, IgG-polymorphisms and post-translational modification of the antibodies in the form of glycosylation, affect IgG-function will be the focus of the current review.

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Spheroid culture as a tool for creating 3D complex tissues.

Eelco M Fennema, Nicolas Rivron, Jeroen Rouwkema … (2013)

3D cell culture methods confer a high degree of clinical and biological relevance to in vitro models. This is specifically the case with the spheroid culture, where a small aggregate of cells grows free of foreign materials. In spheroid cultures, cells secrete the extracellular matrix (ECM) in which they reside, and they can interact with cells from their original microenvironment. The value of spheroid cultures is increasing quickly due to novel microfabricated platforms amenable to high-throughput screening (HTS) and advances in cell culture. Here, we review new possibilities that combine the strengths of spheroid culture with new microenvironment fabrication methods that allow for the creation of large numbers of highly reproducible, complex tissues. Copyright © 2013. Published by Elsevier Ltd.

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Author and article information

Contributors

Sandra Kleinau: Sandra.Kleinau@icm.uu.se

Journal

Journal ID (nlm-ta): BMC Cancer

Journal ID (iso-abbrev): BMC Cancer

Title: BMC Cancer

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2407

Publication date (Electronic): 20 June 2022

Publication date PMC-release: 20 June 2022

Publication date Collection: 2022

Volume: 22

Electronic Location Identifier: 678

Affiliations

[1 ]GRID grid.8993.b, ISNI 0000 0004 1936 9457, Department of Cell and Molecular Biology, , Uppsala University, ; Uppsala, Sweden

[2 ]GRID grid.6612.3, ISNI 0000 0004 1937 0642, Department of Biomedicine, , University of Basel, ; Basel, Switzerland

Article

Publisher ID: 9772

DOI: 10.1186/s12885-022-09772-1

PMC ID: 9210731

PubMed ID: 35725455

SO-VID: 7e867cf2-83df-4dbb-8318-fbbc8be4b25a

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 28 March 2022

Date accepted : 13 June 2022

Funding

Funded by: Uppsala University

Open Access :

Open access funding provided by Uppsala University.

Custom metadata

ScienceOpen disciplines: Oncology & Radiotherapy

Keywords: rituximab,isotypes,b-cell lymphoma,3-dimensional,spheroids,complement-dependent cytotoxicity,cd20,cd59

Data availability:

ScienceOpen disciplines: Oncology & Radiotherapy

Keywords: rituximab, isotypes, b-cell lymphoma, 3-dimensional, spheroids, complement-dependent cytotoxicity, cd20, cd59

Exploring complement-dependent cytotoxicity by rituximab isotypes in 2D and 3D-cultured B-cell lymphoma

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Abstract

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Supplementary Information

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Karger: Oncology

Most cited references 46

Understanding the tumor immune microenvironment (TIME) for effective therapy

IgG Subclasses and Allotypes: From Structure to Effector Functions

Spheroid culture as a tool for creating 3D complex tissues.

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