Complement-dependent cytotoxicity: Difference between revisions
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Bphysrafri (talk | contribs) linked to the classical complement pathway as most important information is there. Rewrote a bit. |
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'''Complement-dependent cytotoxicity''' (CDC) is an effector function of [[Immunoglobulin G|IgG]] and [[Immunoglobulin M|IgM]] [[Antibody|antibodies]]. When they are bound to surface antigen, the [[classical complement pathway]] is triggered, resulting in formation of a [[Complement membrane attack complex|membrane attack complex]] (MAC) and target cell lysis. |
'''Complement-dependent cytotoxicity''' (CDC) is an effector function of [[Immunoglobulin G|IgG]] and [[Immunoglobulin M|IgM]] [[Antibody|antibodies]]. When they are bound to surface [[antigen]] on target cell (e.g. bacterial or viral infected cell), the [[classical complement pathway]] is triggered by bonding [[protein]] [[Complement component 1q|C1q]] to these antibodies, resulting in formation of a [[Complement membrane attack complex|membrane attack complex]] (MAC) and target cell lysis. |
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Complement system is efficiently activated by human IgG1, IgG3 and IgM antibodies, weakly by IgG2 antibodies and it is not activated by IgG4 antibodies.<ref>{{Cite journal|last=Schroeder|first=Harry W.|last2=Cavacini|first2=Lisa|date=2010|title=Structure and function of immunoglobulins|url=http://www.sciencedirect.com/science/article/pii/S0091674909014651|journal=Journal of Allergy and Clinical Immunology|series=2010 Primer on Allergic and Immunologic Diseases|volume=125|issue=2, Supplement 2|pages=S41–S52|doi=10.1016/j.jaci.2009.09.046|issn=0091-6749|via=}}</ref> |
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| ⚫ | It is one mechanism of action by which therapeutic antibodies<ref name="Zhou2008">[http://theoncologist.alphamedpress.org/content/13/9/954.full The Role of Complement in the Mechanism of Action of Rituximab for B-Cell Lymphoma: Implications for Therapy. Zhou 2008]</ref> or [[antibody fragment]]s<ref>[http://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/v15n5-3/1505 Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling.] {{webarchive|url=https://web.archive.org/web/20160409071218/http://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/v15n5-3/1505 |date=2016-04-09 }}</ref> can achieve an antitumor effect.{{ |
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| ⚫ | It is one mechanism of action by which [[Monoclonal antibody therapy|therapeutic antibodies]]<ref name="Zhou2008">[http://theoncologist.alphamedpress.org/content/13/9/954.full The Role of Complement in the Mechanism of Action of Rituximab for B-Cell Lymphoma: Implications for Therapy. Zhou 2008]</ref> or [[antibody fragment]]s<ref>[http://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/v15n5-3/1505 Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling.] {{webarchive|url=https://web.archive.org/web/20160409071218/http://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/v15n5-3/1505 |date=2016-04-09 }}</ref> can achieve an antitumor effect.<ref>{{Cite journal|last=Meyer|first=Saskia|last2=Leusen|first2=Jeanette HW|last3=Boross|first3=Peter|date=2014|title=Regulation of complement and modulation of its activity in monoclonal antibody therapy of cancer|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622586/|journal=mAbs|volume=6|issue=5|pages=1133–1144|doi=10.4161/mabs.29670|issn=1942-0862|pmc=4622586|pmid=25517299|via=}}</ref><ref>{{Cite journal|last=Wang|first=Xinhua|last2=Mathieu|first2=Mary|last3=Brezski|first3=Randall J.|date=2018|title=IgG Fc engineering to modulate antibody effector functions|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777978/|journal=Protein & Cell|volume=9|issue=1|pages=63–73|doi=10.1007/s13238-017-0473-8|issn=1674-800X|pmc=5777978|pmid=28986820|via=}}</ref> |
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==Assays== |
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CDC is measured with various assays to evaluate the effect of drug therapies. |
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== Use of CDC assays == |
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{{expand section|date=February 2016}} |
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=== Therapeutic antibodies === |
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Development of antitumor therapeutic antibodies involves ''[[in vitro]]'' analysis of their effector functions including ability to trigger CDC to kill target cells. Classical approach is to incubate antibodies with target cells and source of complement ([[Serum (blood)|serum]]). Then cell death is determined with several approaches: |
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* '''''Radioactive method'':''' target cells are labeled with [[Chromium-51|<sup>51</sup>Cr]] before CDC assay, chromium is released during cell lysis and amout of [[Radioactive decay|radioactivity]] is measured.<ref name=":0">{{Cite journal|last=Taylor|first=Ronald P.|last2=Lindorfer|first2=Margaret A.|date=2014|title=The role of complement in mAb-based therapies of cancer|url=https://www.ncbi.nlm.nih.gov/pubmed/23886909|journal=Methods (San Diego, Calif.)|volume=65|issue=1|pages=18–27|doi=10.1016/j.ymeth.2013.07.027|issn=1095-9130|pmid=23886909|via=}}</ref><ref name=":1">{{Cite journal|last=Hernandez|first=Axel|last2=Parmentier|first2=Julie|last3=Wang|first3=Youzhen|last4=Cheng|first4=Jane|last5=Bornstein|first5=Gadi Gazit|date=2012|title=Monoclonal antibody lead characterization: in vitro and in vivo methods|url=https://www.ncbi.nlm.nih.gov/pubmed/22907374|journal=Methods in Molecular Biology (Clifton, N.J.)|volume=907|pages=557–594|doi=10.1007/978-1-61779-974-7_32|issn=1940-6029|pmid=22907374}}</ref> |
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* '''''Measuring of the metabolic activity of live cells (live cells staining):''''' after incubation of target cells with antibodies and complement, [[Cell membrane|plasma membrane]]-permeable dye is added (e.g. Calcein AM<ref name=":1" /> or Alamar Blue<ref name=":0" />). Live cells metabolise it into impermeable [[Fluorescence|fluorescent]] product that can be detected by [[flow cytometry]]. This product can’t be formed in metabolically inactive dead cells. |
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* '''''Measuring of the activity of released intracellular enzymes''''': dead cells release [[enzyme]] (e.g. [[Lactate dehydrogenase|LDH]] or [[Glyceraldehyde 3-phosphate dehydrogenase|GAPDH]])<ref name=":0" /> and addition of its [[Substrate (chemistry)|substrate]] leads to color change, that is usually quantified as change of [[absorbance]] or [[Luminescence|luminiscence]]. |
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* '''''Dead cells staining''':'' a (fluorescent) dye gets inside the dead cells through their damaged plasma membrane. For instance [[propidium iodide]] binds to [[DNA]] of dead cells and fluorescent signal is measured by flow cytometry.<ref name=":0" /> |
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=== '''HLA typing and crossmatch test''' === |
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CDC assays are used to find a suitable donor for organ or [[bone marrow]] transplantation, namely donor with matching [[phenotype]] of [[Major histocompatibility complex|histocompatibility system]] [[Human leukocyte antigen|HLA]].<ref name=":2">{{Citation|last=Gautreaux|first=Michael D.|title=Chapter 17 - Histocompatibility Testing in the Transplant Setting|date=2017|url=http://www.sciencedirect.com/science/article/pii/B9780128017340000175|work=Kidney Transplantation, Bioengineering and Regeneration|volume=|pages=223–234|editor-last=Orlando|editor-first=Giuseppe|publisher=Academic Press|isbn=9780128017340|access-date=2019-08-30|editor2-last=Remuzzi|editor2-first=Giuseppe|editor3-last=Williams|editor3-first=David F.}}</ref> At first, HLA typing is done for patient and donor to determine their HLA phenotypes. When potentially suitable couple is found, crossmatch test is done to exclude that patient produces donor-specific anti-HLA antibodies, which could cause [[Transplant rejection|graft rejection]]. |
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CDC form of HLA typing (other words serologic typing) uses batch of anti-HLA antibodies from characterised [[allogeneic]] [[Antiserum|antisera]] or [[Monoclonal antibody|monoclonal antibodies]]. These antibodies are incubated one by one with patient‘s or donor‘s [[Lymphocyte|lymphocytes]] and source of complement. Amount of dead cells (and thus positive result) is measured by dead or live cells staining. Nowadays CDC typing is being replaced by molecular typing, which can identify [[nucleotide]] sequences of HLA molecules via [[Polymerase chain reaction|PCR]].<ref name=":2" /> |
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CDC assay is usually used for performing crossmatch test. The basic version involves incubation of patient’s serum with donor’s lymphocytes and second incubation after adding rabbit complement. Presence of dead cell (positive test) means that donor isn‘t suitable for this particular patient. There are modifications available to increase test sensitivity including extension of minimal incubation time, adding [[Anti Human Globulin|antihuman globulin]] (AHG), removing unbound antibodies before adding complement, separation of [[T cell]] and [[B cell]] subset. Besides CDC crossmatch there is flow-cytometric crossmatch available, that is more sensitive and can detect even complement non-activating antibodies.<ref>{{Cite journal|last=Guillaume|first=Nicolas|date=2018|title=Improved flow cytometry crossmatching in kidney transplantation|url=https://www.ncbi.nlm.nih.gov/pubmed/30270577|journal=HLA|volume=92|issue=6|pages=375–383|doi=10.1111/tan.13403|issn=2059-2310|pmid=30270577|via=}}</ref> |
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==See also== |
==See also== |
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[[Category:Complement system|*]] |
[[Category:Complement system|*]] |
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[[Category:Immunology]] |
[[Category:Immunology]] |
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{{immunology-stub}} |
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Revision as of 14:53, 30 August 2019
Complement-dependent cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When they are bound to surface antigen on target cell (e.g. bacterial or viral infected cell), the classical complement pathway is triggered by bonding protein C1q to these antibodies, resulting in formation of a membrane attack complex (MAC) and target cell lysis.
Complement system is efficiently activated by human IgG1, IgG3 and IgM antibodies, weakly by IgG2 antibodies and it is not activated by IgG4 antibodies.[1]
It is one mechanism of action by which therapeutic antibodies[2] or antibody fragments[3] can achieve an antitumor effect.[4][5]
Use of CDC assays
Therapeutic antibodies
Development of antitumor therapeutic antibodies involves in vitro analysis of their effector functions including ability to trigger CDC to kill target cells. Classical approach is to incubate antibodies with target cells and source of complement (serum). Then cell death is determined with several approaches:
- Radioactive method: target cells are labeled with 51Cr before CDC assay, chromium is released during cell lysis and amout of radioactivity is measured.[6][7]
- Measuring of the metabolic activity of live cells (live cells staining): after incubation of target cells with antibodies and complement, plasma membrane-permeable dye is added (e.g. Calcein AM[7] or Alamar Blue[6]). Live cells metabolise it into impermeable fluorescent product that can be detected by flow cytometry. This product can’t be formed in metabolically inactive dead cells.
- Measuring of the activity of released intracellular enzymes: dead cells release enzyme (e.g. LDH or GAPDH)[6] and addition of its substrate leads to color change, that is usually quantified as change of absorbance or luminiscence.
- Dead cells staining: a (fluorescent) dye gets inside the dead cells through their damaged plasma membrane. For instance propidium iodide binds to DNA of dead cells and fluorescent signal is measured by flow cytometry.[6]
HLA typing and crossmatch test
CDC assays are used to find a suitable donor for organ or bone marrow transplantation, namely donor with matching phenotype of histocompatibility system HLA.[8] At first, HLA typing is done for patient and donor to determine their HLA phenotypes. When potentially suitable couple is found, crossmatch test is done to exclude that patient produces donor-specific anti-HLA antibodies, which could cause graft rejection.
CDC form of HLA typing (other words serologic typing) uses batch of anti-HLA antibodies from characterised allogeneic antisera or monoclonal antibodies. These antibodies are incubated one by one with patient‘s or donor‘s lymphocytes and source of complement. Amount of dead cells (and thus positive result) is measured by dead or live cells staining. Nowadays CDC typing is being replaced by molecular typing, which can identify nucleotide sequences of HLA molecules via PCR.[8]
CDC assay is usually used for performing crossmatch test. The basic version involves incubation of patient’s serum with donor’s lymphocytes and second incubation after adding rabbit complement. Presence of dead cell (positive test) means that donor isn‘t suitable for this particular patient. There are modifications available to increase test sensitivity including extension of minimal incubation time, adding antihuman globulin (AHG), removing unbound antibodies before adding complement, separation of T cell and B cell subset. Besides CDC crossmatch there is flow-cytometric crossmatch available, that is more sensitive and can detect even complement non-activating antibodies.[9]
See also
- Contrast with antibody-dependent cellular cytotoxicity (ADCC)
References
- ^ Schroeder, Harry W.; Cavacini, Lisa (2010). "Structure and function of immunoglobulins". Journal of Allergy and Clinical Immunology. 2010 Primer on Allergic and Immunologic Diseases. 125 (2, Supplement 2): S41–S52. doi:10.1016/j.jaci.2009.09.046. ISSN 0091-6749.
- ^ The Role of Complement in the Mechanism of Action of Rituximab for B-Cell Lymphoma: Implications for Therapy. Zhou 2008
- ^ Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling. Archived 2016-04-09 at the Wayback Machine
- ^ Meyer, Saskia; Leusen, Jeanette HW; Boross, Peter (2014). "Regulation of complement and modulation of its activity in monoclonal antibody therapy of cancer". mAbs. 6 (5): 1133–1144. doi:10.4161/mabs.29670. ISSN 1942-0862. PMC 4622586. PMID 25517299.
- ^ Wang, Xinhua; Mathieu, Mary; Brezski, Randall J. (2018). "IgG Fc engineering to modulate antibody effector functions". Protein & Cell. 9 (1): 63–73. doi:10.1007/s13238-017-0473-8. ISSN 1674-800X. PMC 5777978. PMID 28986820.
- ^ a b c d Taylor, Ronald P.; Lindorfer, Margaret A. (2014). "The role of complement in mAb-based therapies of cancer". Methods (San Diego, Calif.). 65 (1): 18–27. doi:10.1016/j.ymeth.2013.07.027. ISSN 1095-9130. PMID 23886909.
- ^ a b Hernandez, Axel; Parmentier, Julie; Wang, Youzhen; Cheng, Jane; Bornstein, Gadi Gazit (2012). "Monoclonal antibody lead characterization: in vitro and in vivo methods". Methods in Molecular Biology (Clifton, N.J.). 907: 557–594. doi:10.1007/978-1-61779-974-7_32. ISSN 1940-6029. PMID 22907374.
- ^ a b Gautreaux, Michael D. (2017), Orlando, Giuseppe; Remuzzi, Giuseppe; Williams, David F. (eds.), "Chapter 17 - Histocompatibility Testing in the Transplant Setting", Kidney Transplantation, Bioengineering and Regeneration, Academic Press, pp. 223–234, ISBN 9780128017340, retrieved 2019-08-30
- ^ Guillaume, Nicolas (2018). "Improved flow cytometry crossmatching in kidney transplantation". HLA. 92 (6): 375–383. doi:10.1111/tan.13403. ISSN 2059-2310. PMID 30270577.