Apresentação em tema: "What In the World Is a Monoclonal Antibody?. Antibodies x Antigens."— Transcrição da apresentação:
What In the World Is a Monoclonal Antibody?
Antibodies 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 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 Prêmio Nobel - 1984 Georges Köhler (1946-1995) Cesar Milstein (1927-2002)
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
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 - Produção Hybridoma
Diluição Limitante Um clone por poçoUm clone por poço Certeza de que um tipo de clone está proliferando em um poçoCerteza de que um tipo de clone está proliferando em um poço Teste de detecçãoTeste de detecção Diluição limitanteDiluição limitante Teste de detecçãoTeste de detecção Expansão – produção dos AcMoExpansão – produção dos AcMo
Resposta à vacinação
Vias de síntese de nucleotídeos
ELISA Imunofluorescência Indireta Detecção dos clones de interesse
Perform a fusion - Growing the cells (hybridomas) Cells are grown in a 37 o C incubator. Cells are kept in an atmosphere of about 6% CO 2. 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.
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?
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.htmwww.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 CellRadioactive Antibodies
Cancer CellRadioactive 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)
Human MAb production Immune response to mouse antibodies (HAMA response: clearance + disease) HAMA: human anti-mouse antibodies
Anticorpos 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 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 Monoclonais Alternativas Anticorpos monoclonais quiméricos Anticorpos monoclonais humanizados Phage display Camundongos transgênicos
Chimera e Belorofonte
Anticorpos monoclonais quiméricos
Anticorpos monoclonais humanizados
Humanized antibodies Anti-CD20 MAbs (Rituxan) for treatment of non-Hodgkins 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
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
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
http://www.sh.lsuhsc.edu/ intragrad/micro/micro297/4 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.
Unconjugated MAbs as a molecular drugs Target AntigenClinical Use 1. DrugsOverdose 2. TNF- Septic shock 3. VirusesInfection 4. CD3Transplants 5. IL-2RLeukaemia/ lymphoma 6. Anti-venomsSnake 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.