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What In the World Is a
Monoclonal Antibody?
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Antibodies x AntigensAntibodies x Antigens
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Antibodies
Polyclonal Monoclonal
Antibodies that are collected from sera of exposed animal
recognize multiple antigenic sites of injected biochemical.
Individual B lymphocyte hybridoma is cloned and cultured.
Secreted antibodies are collected from culture media
recognize ONE antigenic site
of injected biochemical
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Monoclonal AntibodiesMonoclonal Antibodies
Populações homogêneas de anticorpos derivados Populações homogêneas de anticorpos derivados
de uma única célula produtora de anticorpo, na qual de uma única célula produtora de anticorpo, na qual
todos os anticorpos possuem a mesma todos os anticorpos possuem a mesma
especificidade para um determinante antigênico.especificidade para um determinante antigênico.
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Monoclonal x policlonal antibodiesMonoclonal x policlonal antibodies
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&&
Anticorpos monoclonais -1975Anticorpos monoclonais -1975
Prêmio Nobel - 1984Prêmio Nobel - 1984
Georges KöhlerGeorges Köhler(1946-1995)(1946-1995)
Cesar MilsteinCesar Milstein(1927-(1927-20022002))
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Uses of Monoclonal Antibodies:
• Protein purification• Identification and isolation of cell sub-populations
using fluorescence cell sorting.• Tumor detection and imaging• Tumor killing (magic bullets)• Diagnostic reagents
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How does we make a monoclonal antibody?
• Immunize mice• Test the serum• Perform a fusion• Screen the fusion for the right cells• Grow the hybridomas• Harvest the antibody• Concentrate and purify the product
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IMMUNIZE THE MICE
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IMMUNIZE THE MICE
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Test the serum - Bleeding the mice
• Blood is drawn into the tube.
• A capillary tube is applied to nicked vein.
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Test the serum - Decision time
• When the serum titer of the mice has reached a plateau, an additional ELISA test is performed to determine the predominant isotype present. The two isotypes that are most common in mouse serum are IgG and IgM.
• A fusion is done when the IgG level is high and the IgM level is low.
• Sometimes additional testing is done (Western blots, immunoflurorescence) to determine whether the serum response is specific for the selected antigen.
• The mouse with the strongest, most specific response is chosen for the fusion.
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Anticorpos Monoclonais - ProduçãoAnticorpos Monoclonais - ProduçãoHybridoma
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Diluição LimitanteDiluição Limitante
•Um clone por poçoUm clone por poço
•Certeza de que um tipo de clone está Certeza de que um tipo de clone está proliferando em um poçoproliferando em um poço
•Teste de detecçãoTeste de detecção
•Diluição limitanteDiluição limitante
•Teste de detecçãoTeste de detecção
•Expansão – produção dos AcMoExpansão – produção dos AcMo
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Perform a fusion - Growing the cells (hybridomas)
• Cells are grown in a 37o C incubator.
• Cells are kept in an atmosphere of about 6% CO2.
• The cells are fed after 7 days of incubation.
• The cells are checked for growth after 10 days of incubation.
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Harvest the antibody
1) The antibody is produced by the cells and released into the cell supernatant.
2) Media is added to the flasks until the desired volume of antibody-containing supernatant is obtained.
3) When the desired volume is reached, the supernatant is refrigerated and tested by ELISA.
4) If the testing is successful, the supernatant can be used by the investigator, or concentrated and purified.
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Expand in mice or expand in vitro
in vitro material is less concentrated and contains bovine serum
ascites fluid contain high [mAb]
and minor contaminationwith mouse Ig
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Concentrate and Purify the Product
• The antibody can be concentrated by precipitation with a solution of saturated ammonium sulfate.
• Concentrated antibody can then be purified by passing it through a column.
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Monoclonal Antibodies Monoclonal Antibodies
2005: 37.934 articles
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Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades
Imunodiagnóstico/Sorologia Imunodiagnóstico/Sorologia
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Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades
SorotipagemSorotipagem
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Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades
Imunologia CelularImunologia Celular
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Subpopulations of cells can by physically separated
using antibodies coupled to magnetic beads
Dynabeads:
T Cells
B Cells
NK Cells
Stem Cells
Monocytes
Dendritic Cells
Granulocytes
Endothelial Cells
Leucocytes
Tumour Cells
Which human cell type do you want to isolate?
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Miosina
Actina
Tubulina
Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades
Biologia CelularBiologia Celular
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Human MAb production
Immune response to mouse antibodies
(HAMA response: clearance + disease)HAMA: human anti-mouse antibodies
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Anticorpos monoclonais humanosAnticorpos monoclonais humanos
DIFICULDADES (mielomas):
Falta de linhagens de células de mieloma humano bem estabelecida
Mieloma secretores de Ig
Crescimento deficiente
Human MAb production
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Anticorpos monoclonais humanosAnticorpos monoclonais humanos
DIFICULDADES (linfócitos B):
Obtenção das células B antígenos-específicas ativadas
sangue x baço/linfonodos
Alternativa: imunizar indivíduos antes de retirar os linf. B
Estimular células B in vitro com Ag
produção de IgM de baixa afinidade
Transferência de linfócitos humanos para SCDI mice
Transformação de linfócitos B com Epstein-Barr
Human MAb production
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Produção de Anticorpos MonoclonaisProdução de Anticorpos Monoclonais
AlternativasAlternativas
•Anticorpos monoclonais quiméricos
•Anticorpos monoclonais humanizados
•Phage display
•Camundongos transgênicos
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ChimeraChimera
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Chimera e BelorofonteChimera e Belorofonte
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Anticorpos monoclonais quiméricosAnticorpos monoclonais quiméricos
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Anticorpos monoclonais quiméricosAnticorpos monoclonais quiméricos
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Anticorpos monoclonais Anticorpos monoclonais humanizadoshumanizados
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Humanized antibodies
Anti-CD20 MAbs (Rituxan) for treatment of non-Hodgkin’s lymphoma
Mouse antigen-interacting sequences
Clone those genes and express them
as a recombinant protein in the protein expression system
Sales of Rituxan/MabThera by Year
http://www.sh.lsuhsc.edu/intragrad/micro/micro297/4
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Phage displayPhage display
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Single chain Fv fragments
http://www.gene-therapeutics.com/Service/custom%20scFv%20antibodies/Abb-scFv.gif
The size and specificity of the ScFv fragment
may allow for attachment to cryptic sites
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Phage displayPhage display
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Phage displayPhage display
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Phage displayPhage display
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the enrichment of an antigen-specific
phage-antibody (circle) on a background of
non specific phage-antibodies (square).
Selection of antibodies from phage display libraries:
Antigen comes from serum of cancer patients
www.pharma.ethz.ch/bmm/ protocols/schema.jpg
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Transgenic miceTransgenic mice
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http://www.sh.lsuhsc.edu/intragrad/micro/micro297/4
Xenomouse(mouse with human immunoglobulin loci)
Abgenix
XenoMouse genetically engineeredTo replace murine IgH and Igk loci
with their human Ig counterparts on YACHuman Ig transgenes
carry the majorityof the human variable repertoire
and can undergoclass switching
from IgM to IgG isotypes.
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Monoclonal Antibody with Cancer Cell
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Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades
ImunoterapiaImunoterapia
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Immunotoxin: Antibody-Toxin Conjugate
waynesword.palomar.edu/ images/antibod2.gif
ricin from the castor bean (Ricinus communis).
Antibody is tumor-specific
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Only the targeted tumor cells are killed by the toxin
Common side effect –Vascular leak syndrome.
Could be relieved by usage of mutated toxin
Bunch of other toxins could be used
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Saponin (from soapwort)
: www.som.soton.ac.uk/.../members/ DFlavell/djf2.htm
potent single chain ribosome inactivating protein
Goal: treatment of leukemias
and lymphomas
For T-cell leukemia could be targeted via CD7
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How Radioimmunotherapy Works
Cancer Cell Radioactive Antibodies
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Cancer Cell Radioactive Antibodies
How Radioimmunotherapy Works
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OctreoScan in a patient with metastatic small cell carcinoma of the prostate
www.stjosephs-marshfield.org/
Monoclonal antibodies, which are specific for a certain antigen, i.e. prostate carcinoma cells, can be tagged with a radioactive tracer,
which when injected into the body will localize in areas of these recurrent carcinoma cells
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FDA Approved RIT
2 current FDA-approved RIT drugs:
– Zevalin™ (2002)
– Bexxar™ (2003)
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Unconjugated MAbs as a molecular drugs
Target Antigen Clinical Use1. Drugs Overdose2. TNF- Septic shock3. Viruses Infection4. CD3 Transplants5. IL-2R Leukaemia/
lymphoma6. Anti-venoms Snake and Bee Bites
Organ Transplants:Donor T cells cause graft vs host (GvH) reaction.Donor T cells eliminated using anti-CD3 Mabs (OKT3) Muromonab; .Treat patients with anti-CD3 after solid organ transplants.
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http://www.roche.com/pages/facets/9/herc1.jpg
Herceptin (TrastuzuMAb) anti-HER MAbs
Approved!
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Comercial chimeric and humanized Comercial chimeric and humanized antibodiesantibodies
2003: 9 AcMo quiméricos ou humanizados aprovados pelo FDA
Leucemia, linfoma não-Hodgkin, rejeição de órgãos, doenças auto-imunes e câncer de mama
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Comercial chimeric and humanized Comercial chimeric and humanized antibodiesantibodies
Autoimmune diseaseAutoimmune disease
RA: artrite reumatóide; MS: esclerose múltipla
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Comercial human monoclonal Comercial human monoclonal antibodiesantibodies
Autoimmune diseaseAutoimmune disease
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Want to know more? Just ask!