What In the World Is a

Monoclonal Antibody?

Antibodies x AntigensAntibodies x Antigens

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

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.

Monoclonal x policlonal antibodiesMonoclonal x policlonal antibodies

&&

Anticorpos monoclonais -1975Anticorpos monoclonais -1975

Prêmio Nobel - 1984Prêmio Nobel - 1984

Georges KöhlerGeorges Köhler(1946-1995)(1946-1995)

Cesar MilsteinCesar Milstein(1927-(1927-20022002))

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

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

IMMUNIZE THE MICE

IMMUNIZE THE MICE

Test the serum - Bleeding the mice

• Blood is drawn into the tube.

• A capillary tube is applied to nicked vein.

Test the serum – In the lab

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.

Anticorpos Monoclonais - ProduçãoAnticorpos Monoclonais - ProduçãoHybridoma

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

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.

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

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.

Monoclonal Antibodies Monoclonal Antibodies

2005: 37.934 articles

Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades

Imunodiagnóstico/Sorologia Imunodiagnóstico/Sorologia

Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades

SorotipagemSorotipagem

Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades

Imunologia CelularImunologia Celular

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?

Miosina

Actina

Tubulina

Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades

Biologia CelularBiologia Celular

Human MAb production

Immune response to mouse antibodies

(HAMA response: clearance + disease)HAMA: human anti-mouse antibodies

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

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

Produção de Anticorpos MonoclonaisProdução de Anticorpos Monoclonais

AlternativasAlternativas

•Anticorpos monoclonais quiméricos

•Anticorpos monoclonais humanizados

•Phage display

•Camundongos transgênicos

ChimeraChimera

Chimera e BelorofonteChimera e Belorofonte

Anticorpos monoclonais quiméricosAnticorpos monoclonais quiméricos

Anticorpos monoclonais quiméricosAnticorpos monoclonais quiméricos

Anticorpos monoclonais Anticorpos monoclonais humanizadoshumanizados

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

Phage displayPhage display

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

Phage displayPhage display

Phage displayPhage display

Phage displayPhage display

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

Transgenic miceTransgenic mice

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.

Monoclonal Antibody with Cancer Cell

Anticorpos Monoclonais - UtilidadesAnticorpos Monoclonais - Utilidades

ImunoterapiaImunoterapia

Immunotoxin: Antibody-Toxin Conjugate

waynesword.palomar.edu/ images/antibod2.gif

ricin from the castor bean (Ricinus communis).

Antibody is tumor-specific

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

Goal: treatment of leukemias

and lymphomas

For T-cell leukemia could be targeted via CD7

How Radioimmunotherapy Works

Cancer Cell Radioactive Antibodies

Cancer Cell Radioactive Antibodies

How Radioimmunotherapy Works

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

FDA Approved RIT

2 current FDA-approved RIT drugs:

– Zevalin™ (2002)

– Bexxar™ (2003)

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.

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

Herceptin (TrastuzuMAb) anti-HER MAbs

Approved!

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

Comercial chimeric and humanized Comercial chimeric and humanized antibodiesantibodies

Autoimmune diseaseAutoimmune disease

RA: artrite reumatóide; MS: esclerose múltipla

Comercial human monoclonal Comercial human monoclonal antibodiesantibodies

Autoimmune diseaseAutoimmune disease

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