What In the World Is a Monoclonal Antibody?

Antibodies x Antigens

Antibodies Polyclonal Monoclonal recognize multiple antigenic sites 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

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.

Monoclonal x policlonal antibodies

Anticorpos monoclonais -1975 & 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

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 A capillary tube is applied to nicked vein. Blood is drawn into the tube.

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 - Produção Hybridoma Anticorpos Monoclonais - Produção

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

Resposta à vacinação

Vias de síntese de nucleotídeos

Detecção dos clones de interesse Imunofluorescência Indireta ELISA

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 contamination with 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 2005: 37.934 articles

Anticorpos Monoclonais - Utilidades Imunodiagnóstico/Sorologia

Anticorpos Monoclonais - Utilidades Sorotipagem

Anticorpos Monoclonais - Utilidades Imunologia Celular

Dynabeads: Subpopulations of cells can by physically separated using antibodies coupled to magnetic beads Dynabeads: Which human cell type do you want to isolate? T Cells B Cells NK Cells Stem Cells Monocytes Dendritic Cells Granulocytes Endothelial Cells Leucocytes Tumour Cells

Anticorpos Monoclonais - Utilidades Biologia Celular Miosina Actina Tubulina

Human MAb production Immune response to mouse antibodies (HAMA response: clearance + disease) HAMA: human anti-mouse antibodies

Anticorpos monoclonais humanos DIFICULDADES (mielomas): Human MAb production Anticorpos monoclonais humanos DIFICULDADES (mielomas): Falta de linhagens de células de mieloma humano bem estabelecida Mieloma secretores de Ig Crescimento deficiente

Anticorpos monoclonais humanos DIFICULDADES (linfócitos B): 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

Produção de Anticorpos Monoclonais Alternativas Anticorpos monoclonais quiméricos Anticorpos monoclonais humanizados Phage display Camundongos transgênicos

Chimera

Chimera e Belorofonte

Anticorpos monoclonais quiméricos

Anticorpos monoclonais quiméricos

Anticorpos monoclonais humanizados

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

Phage display

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

Phage display

Phage display

Phage display

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). www.pharma.ethz.ch/bmm/ protocols/schema.jpg Antigen comes from serum of cancer patients

Transgenic mice

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

Monoclonal Antibody with Cancer Cell

Anticorpos Monoclonais - Utilidades Imunoterapia

Immunotoxin: Antibody-Toxin Conjugate is tumor-specific ricin from the castor bean (Ricinus communis). waynesword.palomar.edu/ images/antibod2.gif

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) potent single chain ribosome inactivating protein                                                                                                                Goal: treatment of leukemias and lymphomas For T-cell leukemia could be targeted via CD7 : www.som.soton.ac.uk/.../members/ DFlavell/djf2.htm

How Radioimmunotherapy Works Cancer Cell Radioactive Antibodies

Radioactive Antibodies How Radioimmunotherapy Works Cancer Cell Radioactive Antibodies

Monoclonal antibodies, which are specific for a certain antigen, i. e 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)

Unconjugated MAbs as a molecular drugs Target Antigen Clinical Use 1. Drugs Overdose 2. TNF- Septic shock 3. Viruses Infection 4. CD3 Transplants 5. IL-2R Leukaemia/ lymphoma 6. 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.

Herceptin (TrastuzuMAb) anti-HER MAbs Approved! http://www.roche.com/pages/facets/9/herc1.jpg

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

Comercial chimeric and humanized antibodies Autoimmune disease RA: artrite reumatóide; MS: esclerose múltipla

Comercial human monoclonal antibodies Autoimmune disease

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