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What In the World Is a Monoclonal Antibody?. Antibodies x Antigens. 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.

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antibodies
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

slide4

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.

slide6

&

Georges Köhler

(1946-1995)

Cesar Milstein

(1927-2002)

Anticorpos monoclonais -1975

Prêmio Nobel - 1984

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

Uses of Monoclonal Antibodies:
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

How does we make a monoclonal antibody?
test the serum bleeding the mice
Blood is drawn into the tube.

A capillary tube is applied to nicked vein.

Test the serum - Bleeding the mice
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.

Test the serum - Decision time
slide14

Hybridoma

Anticorpos Monoclonais - Produção

slide16

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
perform a fusion growing the cells hybridomas
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.
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.

Harvest the antibody
expand in mice or expand in vitro
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

concentrate and purify the product
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.
slide21

Monoclonal Antibodies

2005: 37.934 articles

slide22

Anticorpos Monoclonais - Utilidades

Imunodiagnóstico/Sorologia

slide25

Subpopulations of cells

can by physically separated

using antibodies

coupled to magnetic beads

Dynabeads:

Which human cell type

do you want to isolate?

slide26

Tubulina

Miosina

Actina

Anticorpos Monoclonais - Utilidades

Biologia Celular

immunotoxin antibody toxin conjugate

waynesword.palomar.edu/ images/antibod2.gif

Immunotoxin: Antibody-Toxin Conjugate

Antibody

is tumor-specific

ricin from the castor bean

(Ricinus communis).

only the targeted tumor cells are killed by the toxin
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

saponin from soapwort

: www.som.soton.ac.uk/.../members/ DFlavell/djf2.htm

potent

single chain ribosome inactivating protein

Saponin (from soapwort)

Goal:

treatment of

leukemias

and lymphomas

For T-cell leukemia could be

targeted via CD7

how radioimmunotherapy works
How Radioimmunotherapy Works

Cancer Cell

RadioactiveAntibodies

slide33

How Radioimmunotherapy Works

Cancer Cell

Radioactive Antibodies

slide34

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

fda approved rit
2 current FDA-approved RIT drugs:

Zevalin™ (2002)

Bexxar™ (2003)

FDA Approved RIT
human mab production
Immune response to mouse antibodies

(HAMA response: clearance + disease)

HAMA: human anti-mouse antibodies

Human MAb production
slide37

Human MAb production

Anticorpos monoclonais humanos

DIFICULDADES (mielomas):

Falta de linhagens de células de mieloma humano bem estabelecida

Mieloma secretores de Ig

Crescimento deficiente

slide38

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

Transferência de linfócitos humanos para SCDI mice

Transformação de linfócitos B com Epstein-Barr

slide39

Produção de Anticorpos Monoclonais

Alternativas

  • Anticorpos monoclonais quiméricos
  • Anticorpos monoclonais humanizados
  • Phage display
  • Camundongos transgênicos
slide46

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-Hodgkin’s lymphoma

single chain fv fragments
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/custom%20scFv%20antibodies/Abb-scFv.gif

selection of antibodies from phage display libraries

www.pharma.ethz.ch/bmm/

protocols/schema.jpg

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

xenomouse mouse with human immunoglobulin loci
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

unconjugated mabs as a molecular drugs
Target AntigenClinical 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.

Unconjugated MAbs as a molecular drugs
herceptin trastuzumab anti her mabs
Herceptin (TrastuzuMAb) anti-HER MAbs

Approved!

http://www.roche.com/pages/facets/9/herc1.jpg

slide57

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

slide58

Comercial chimeric and humanized antibodies

Autoimmune disease

RA: artrite reumatóide; MS: esclerose múltipla