What In the World Is a Monoclonal Antibody?

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• What In the World Is a Monoclonal Antibody?

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Antibodies x Antigens
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Antibodies
Polyclonal
Monoclonal
Individual B lymphocyte hybridoma is cloned and
cultured. Secreted antibodies are collected
from culture media
Antibodies that are collected from sera of
exposed animal
recognize multiple antigenic sites of injected
biochemical.
recognize ONE antigenic site of injected
biochemical
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Monoclonal Antibodies
Populações homogêneas de anticorpos derivados de
uma única célula produtora de anticorpo, na qual
todos os anticorpos possuem a mesma
especificidade para um determinante antigênico.
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Monoclonal x policlonal antibodies
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Georges Köhler (1946-1995)
Cesar Milstein (1927-2002)
Anticorpos monoclonais -1975 Prêmio Nobel - 1984
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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 In the lab
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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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Hybridoma
Anticorpos Monoclonais - Produção
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Diluição Limitante

• Um clone por poço
• Certeza de que um tipo de clone está proliferando
  em um poço
• Teste de detecção
• Diluição limitante
• Teste de detecção
• Expansão produção dos AcMo

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Resposta à vacinação
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Vias de síntese de nucleotídeos
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Detecção dos clones de interesse
Imunofluorescência Indireta
ELISA
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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
contamination with 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
2005 37.934 articles
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Anticorpos Monoclonais - Utilidades Imunodiagnósti
co/Sorologia
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Anticorpos Monoclonais - Utilidades Sorotipagem
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Anticorpos Monoclonais - Utilidades Imunologia
Celular
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Subpopulations of cells can by physically
separated using antibodies coupled to magnetic
beads
Dynabeads
Which human cell type do you want to isolate?
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Anticorpos Monoclonais - Utilidades Biologia
Celular
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Monoclonal Antibody with Cancer Cell
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Anticorpos Monoclonais - Utilidades Imunoterapia
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Immunotoxin Antibody-Toxin Conjugate
Antibody is tumor-specific
ricin from the castor bean (Ricinus communis).
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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)
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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How Radioimmunotherapy Works
Cancer Cell
Radioactive Antibodies
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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
www.stjosephs-marshfield.org/
OctreoScan in a patient with metastatic small
cell carcinoma of the prostate
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FDA Approved RIT

• 2 current FDA-approved RIT drugs
• Zevalin (2002)
• Bexxar (2003)

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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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Human MAb production
Anticorpos monoclonais humanos
DIFICULDADES (mielomas) Falta de linhagens de
células de mieloma humano bem estabelecida Mieloma
secretores de Ig Crescimento deficiente
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Human MAb production
Anticorpos 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 Transfe
rência de linfócitos humanos para SCDI mice
Transformação de linfócitos B com
Epstein-Barr
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Produção de Anticorpos Monoclonais Alternativas

• Anticorpos monoclonais quiméricos
• Anticorpos monoclonais humanizados
• Phage display
• Camundongos transgênicos

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Chimera
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Chimera e Belorofonte
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Anticorpos monoclonais quiméricos
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Anticorpos monoclonais quiméricos
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Anticorpos monoclonais humanizados
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Humanized antibodies
Clone those genes and express them as a
recombinant protein in the protein expression
system
Sales of Rituxan/ MabThera by Year
Mouse antigen-interacting sequences
http//www.sh.lsuhsc.edu/ intragrad/micro/micro297
/4
Anti-CD20 MAbs (Rituxan) for treatment of
non-Hodgkins lymphoma
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Phage display
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Single chain Fv fragments
The size and specificity of the ScFv fragment
may allow for attachment to cryptic sites
http//www.gene-therapeutics.com/Service/custom20
scFv20antibodies/Abb-scFv.gif
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Phage display
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Phage display
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Phage display
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Selection of antibodies from phage display
libraries
the enrichment of an antigen-specific
phage-antibody (circle) on a background of non
specific phage-antibodies (square).
Antigen comes from serum of cancer patients
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Transgenic mice
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Xenomouse(mouse with human immunoglobulin loci)
Abgenix
XenoMouse genetically engineered To replace
murine IgH and Igk loci with their human Ig
counterparts on YAC Human Ig transgenes carry
the majority of the human variable repertoire
and can undergo class switching from IgM to IgG
isotypes.
http//www.sh.lsuhsc.edu/ intragrad/micro/micro297
/4
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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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Herceptin (TrastuzuMAb) anti-HER MAbs
Approved!
http//www.roche.com/pages/facets/9/herc1.jpg
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Comercial chimeric and humanized antibodies
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
antibodies Autoimmune disease
RA artrite reumatóide MS esclerose múltipla
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Comercial human monoclonal antibodies Autoimmune
disease
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