1. What In the World Is a Monoclonal Antibody?

2. Antibodies x Antigens

3. 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

4. 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.

5. Monoclonal x policlonal antibodies

6. & Georges Köhler (1946-1995) Cesar Milstein (1927-2002) Anticorpos monoclonais -1975 Prêmio Nobel - 1984

7. 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:

8. 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?

9. IMMUNIZE THE MICE

10. IMMUNIZE THE MICE

11. Blood is drawn into the tube. A capillary tube is applied to nicked vein. Test the serum - Bleeding the mice

12. Test the serum – In the lab

13. 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

14. Hybridoma Anticorpos Monoclonais - Produção

16. 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

17. 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.

18. 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

19. 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

20. 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.

21. Monoclonal Antibodies 2005: 37.934 articles

22. Anticorpos Monoclonais - Utilidades Imunodiagnóstico/Sorologia

23. Anticorpos Monoclonais - Utilidades Sorotipagem

24. Anticorpos Monoclonais - Utilidades Imunologia Celular

25. Subpopulations of cells can by physically separated using antibodies coupled to magnetic beads Dynabeads: Which human cell type do you want to isolate?

26. Tubulina Miosina Actina Anticorpos Monoclonais - Utilidades Biologia Celular

27. Monoclonal Antibody with Cancer Cell

28. Anticorpos Monoclonais - Utilidades Imunoterapia

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

30. 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

31. : 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

32. How Radioimmunotherapy Works Cancer Cell RadioactiveAntibodies

33. How Radioimmunotherapy Works Cancer Cell Radioactive Antibodies

34. 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

35. 2 current FDA-approved RIT drugs: Zevalin™ (2002) Bexxar™ (2003) FDA Approved RIT

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

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

38. 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

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

40. Chimera

41. Chimera e Belorofonte

42. Anticorpos monoclonais quiméricos

43. Anticorpos monoclonais quiméricos

44. Anticorpos monoclonais humanizados

46. 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

47. Phage display

48. 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

49. Phage display

50. Phage display