Quantifying Antibody Responses Induced by Antigen-Agnostic Immunotherapies

As the development and clinical application of cancer immunotherapies continue to expand, so does the need for novel methods to dissect their mechanisms of action. Antibodies are important effector molecules in cancer therapies due to their potential to bind directly to surface-expressed antigens and facilitate Fc receptor-mediated uptake of antigens by antigen-presenting cells. Quantifying antibodies that are specific for defined antigens is straightforward. However, we describe herein a preclinical method to evaluate tumor-associated and virus-specific antibody responses to antigen-agnostic immunotherapies. This method uses autologous tumor cells as reservoirs of bulk tumor antigens, which can be bound by antibodies from the serum or plasma of tumor-bearing mice. These antibodies can then be detected and quantified using isotype-specific secondary antibodies conjugated to a fluorochrome. Alternatively, virus-infected cells can be used as a source of viral antigens. This method will enable researchers to assess antibody responses following immunotherapies without requiring pre-defined antigens. Alternatively, total virus-specific antibody responses could be studied as an alternative to more limited virus-neutralizing antibody assays. Therefore, this method can facilitate studying the role of humoral responses in the context of immunotherapies, including those that rely on the use of viral vectors. As the development and clinical application of cancer immunotherapies continue to expand, so does the need for novel methods to dissect their mechanisms of action. Antibodies are important effector molecules in cancer therapies due to their potential to bind directly to surface-expressed antigens and facilitate Fc receptor-mediated uptake of antigens by antigen-presenting cells. Quantifying antibodies that are specific for defined antigens is straightforward. However, we describe herein a preclinical method to evaluate tumor-associated and virus-specific antibody responses to antigen-agnostic immunotherapies. This method uses autologous tumor cells as reservoirs of bulk tumor antigens, which can be bound by antibodies from the serum or plasma of tumor-bearing mice. These antibodies can then be detected and quantified using isotype-specific secondary antibodies conjugated to a fluorochrome. Alternatively, virus-infected cells can be used as a source of viral antigens. This method will enable researchers to assess antibody responses following immunotherapies without requiring pre-defined antigens. Alternatively, total virus-specific antibody responses could be studied as an alternative to more limited virus-neutralizing antibody assays. Therefore, this method can facilitate studying the role of humoral responses in the context of immunotherapies, including those that rely on the use of viral vectors. 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Ho T.S. et al.Genetic basis for clinical response to CTLA-4 blockade in melanoma.N. Engl. J. Med. 2014; 371: 2189-2199Crossref PubMed Scopus (3040) Google Scholar We recently developed a method to detect tumor-specific T cell responses to antigen-agnostic immunotherapies.16van Vloten J.P. Santry L.A. McAusland T.M. Karimi K. McFadden G. Petrik J.J. Wootton S.K. Bridle B.W. Quantifying Antigen-Specific T Cell Responses When Using Antigen-Agnostic Immunotherapies.Mol. Ther. Methods Clin. Dev. 2019; 13: 154-166Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar This method relies on the presentation of bulk tumor antigens in the context of major histocompatibility complexes on cancer cells to T cells ex vivo, which can be quantified by flow cytometry. We reasoned that the principle of using cancer cells as targets to detect tumor antigen-specific T cells following the use of antigen-agnostic cancer immunotherapies could be extended to the detection of tumor-associated antibodies. We describe herein a method similar to the in-cell western blot, which uses autologous tumor cells as reservoirs of bulk tumor antigens to bind to serum- or plasma-derived antibodies that can subsequently be detected using species- and isotype-specific fluorochrome-conjugated antibodies for quantification via assessment of relative fluorescence intensity. This method can also be applied to detecting virus-specific antibodies, which is relevant to any treatment that relies on the use of viruses, such as oncolytic virotherapy. This provides a valuable method that can be added to the toolbox of preclinical cancer researchers to evaluate the role of endogenous antibodies induced by antigen-agnostic immunotherapies and can help inform the design of future cancer therapies. •Retro-Orbital Blood Draw○Heparinized microhematocrit capillary tubes (Fisher Scientific, MA, USA, catalog number [Cat#]22-362-566). This allows for a separate assessment of cells, such as T cell responses; alternatively, clotted blood can be used, but this would restrict analysis to serum-derived factors only.○1.5 mL microtubes○Gauze pads○Eye lubricant○Container filled with ice pellets•Cell Culture○Complete DMEM (Fisher Scientific, Cat#SH30022.01) or media specific to the tumor cell line of interest○10% fetal bovine serum (VWR, PA, USA, Cat#97068-085)○Penicillin streptomycin cocktail (Fisher Scientific, Cat#SV30010)○0.25% EDTA (Corning, NY, USA, reference number [Ref#]25-052-CI)○PBS (Fisher Scientific, Cat#SH30256.01)○Cell culture-treated flasks and plates, including 96-well flat-bottom plates○Fibronectic-collagen (FNC) coating mix (AthenaES, MD, USA, Cat#0407)•Sample Processing○HBSS (Fisher Scientific, Cat#SH3003103)○4% Paraformaldehyde (Fisher Scientific, Cat#J19943-K2)○0.2% Triton X-100 (Sigma-Aldrich, MA, USA, Cat#T8787)○BSA (Fisher Scientific, Cat#BP1600100)•Antibody○Goat anti-mouse IgG (H+L)-Alexa Fluor 488 (Fisher Scientific, Cat#A28175). An antibody bound to a different fluorochrome could be used, as long as it can be detected by a plate reader. Also, a different isotype-specific antibody could be used, depending on the isotype(s) of interest. •Plate reader capable of detecting fluorescence at a wavelength of 490 nm. Refer to Figure 1.1.Cell Preparation•Seed 10,000 healthy target tumor cells per well in a 96-well plate.○Target cells should be 100% confluent in each well upon starting the assay. This ensures a high quality signal and prevents binding of the secondary detection antibody to the surface of the plate.•Incubate target cells overnight at 37°C, 5% CO2, and 21% O2.2.Collection of Plasma•Collect blood from mice in 1.5-mL microfuge tubes.○Collecting ∼200 μL blood will yield ∼50 μL plasma for analysis.○Maximizing the blood volume will maximize the sensitivity of the assay but adhere to institutional guidelines. The work presented here was approved by the University of Guelph Animal Care Committee and adhered to the policies published by the Canadian Council on Animal Care.•Place tube containing blood on ice until sample collection is complete.•Centrifuge blood at 500 × g for 10 min at 4°C.•Centrifuged samples will separate plasma or serum to the top layer. Collect clear plasma or serum without disturbing the cellular component beneath and aliquot into new tubes.•Pause point: plasma or serum samples can be stored long-term at −80°C or can continue to assay.3.In-Cell Western Assay•Remove media from 96-well plate and wash cells two times with 100 μL HBSS+Mg2+.•Fix cells with 25 μL 3.7% paraformaldehyde, incubating for 10–15 minutes at room temperature.•Wash cells three times in μL cells with μL Triton incubating for 10 min at room can be the to target antibodies against surface cells three times in μL cells with in μL total volume of incubating for at room point: can be overnight at plasma or serum on ice of plasma or serum samples in in a 96-well plate for and of should be tested for each to that is will depending on the of antibodies induced by a with more potent therapies requiring a the μL plasma or serum samples to target plasma or serum to of target cells. This will as a secondary for at room point: with plasma or serum can be extended to overnight at plasma or serum from and wash three times with μL to the secondary anti-mouse to in μL secondary detection antibody to each well the of that with the secondary antibody and for at room in the the secondary detection antibody and wash three times with μL fluorescence using a plate reader with a 490 the quality of antibody detection using a should have to samples for which antibody responses are should have an of cells with to Figure can be as and then used to the under the the from the on a from with the secondary antibody define the signal and can be from the fluorescence of can be as under the following of the or as the in under the of mice to for the Western from Tumor from and Figure to antibody of antibody responses can be using this method by blood on we 10 and of target to 100% should be or serum and and binding of plasma or serum-derived antibodies: with a secondary from plasma or serum collection to the of this a total of three potential However, the in-cell western assay should be for The of this assay on the use of cells that as reservoirs of target antigens. 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Petrik J. with an Immunotherapy in a of Cancer 2019; PubMed Scopus Google Scholar following tumor we mice with an as virus C.G. M.M. C.T. Bridle B.W. M. et a novel oncolytic and immune Ther. 20: Full Text Full Text PDF PubMed Scopus Google that was into the following blood was and plasma for the in-cell western assay. cells used as targets and plasma samples following the in Figure samples the in-cell western in three with each assay containing secondary samples from tumor-bearing mice that following of mice with the of antibody responses. in to was by These and mice that the in-cell western assay could detect in the antibody following of an immunotherapy to a humoral is important to that these antibody responses or and as responses against antigens with cells is common with many antigen-agnostic the potential of using the in-cell western assay to assess the of an antibody response, mice with cells to following tumor mice with an immunotherapy in the of an cell vaccine blood collection is a plasma could be from mice 10 and following cells used as target cells, and plasma samples in three separate in-cell western assays. with the tumor-associated plasma antibody responses to mice at 10 which by following tumor-bearing mice to the blood therefore to these at the This the in-cell western assay could be applied to the of antibody responses induced by cancer immunotherapies. to the immunogenic of viruses, therapies potent virus-specific immune responses. We reasoned that using cells with the would enable the in-cell western to be applied to detecting virus-specific antibodies. mice with following tumor with plasma 10 following of cells used as cells, as should antigens with tumor cells. cells and to adhere with at an of 10 for We reasoned that this would cell to the virus to maximize of viral without killing a number of cells. The in-cell western assay was then as for the of cells as a with potent virus-specific antibody as by an in to plasma from A for virus-specific antibody responses is by virus is by of cells with antibodies in serum or can be by or flow B. and use of a virus assay on flow detection of PubMed Scopus Google Scholar, W. of and flow methods for virus Clin. 2007; PubMed Scopus Google Scholar However, the of antibodies in to viral by would be detected by is well V. M.A. L.A. K. J.M. D. J. E.J. T cells, antibodies, and is important for virus PubMed Scopus Google Scholar, D.J. P. Wilson antibodies Fc for in Clin. Invest. 2016; PubMed Scopus Google Scholar, G. J. S. M.M. S. B. of are by and 2010; PubMed Scopus Google Scholar, D.M. A. L. A novel role for antibodies against in to Immunol. PubMed Scopus Google Scholar The in-cell western is limited to detecting antibodies, as both and virus would be by target cells. The application of this to detecting virus-specific antibodies will researchers with a to antibody responses to the of viral antigens, which can be with to antibodies. this provides a for detecting tumor-associated antibody responses following antigen-agnostic immunotherapies. the in-cell western method could be applied to any antigen-agnostic therapy capable of tumor-associated antibodies, but limited and therapies that immunogenic cancer cell and should be to any tumor tumor cells as reservoirs of target antigens relevant tumor in the is Therefore, this method could also be for following antigen-specific immunotherapies. we that this method can also as an alternative to other for antibody responses to pre-defined tumor antigens by the target in a cell line that of the The presented herein the antibody detection method to responses to which are to be of This method would also be for detecting secondary antibody for in patients that have tumor-associated antibodies at the of treatment or that of Blood collection is so antibody responses can be in the and to clinically relevant such as The presented here has been for tumor cell in could be applied to cancer cells that in the application to such as flow to be used to studies of tumor-associated or virus-specific antibody responses can be using secondary detection antibodies against other The studies herein on detection of but detection of other such as or could be and used to and immune response the of the antibody the major for this is the existence of a target tumor cell we that this method could be applied For tumor cells could be from patients and to cell ex to facilitate this would be for therapies such as cell tumor cell would then be applied in the method presented herein to antibody responses. we studies to the potential of this we cancer cell from from two both this was in This would facilitate the assessment of antibody responses at or their and and of and of and and of and of the and The This was by a and a from the and an from the of in for Cancer to and an that was by the Canadian Cancer and Canadian of of Cancer to and from also by a from the and Council of was by a Canadian and to and an to We Animal University of for

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