Exploration of Monoclonal Antibodies in Healthcare Innovations

2. CELL Results: 6.591.934 • DNA Results: 1.594.619 Antibody 1.333.661

3. Il mercato dei farmaci 15 anni fa, nel 2004, le vendite di farmaci sul mercato mondiale hanno fatto registrare un incremento del 7%, determinando una spesa complessiva di 550 miliardi di dollari. For the first time ever, in 2006, global spending on prescription drugs topped $643 billion Spending on pharmaceutical drugs will top $1 trillion in 2014 for the first time, The increased spending is led by the introduction of more innovative drugs and expanded access to healthcare in emerging markets led by China. IMS predicts spending will grow at a 3- to 6 percent compound annual rate over the next five years. In addition, by 2017 the United States, France, Germany, the UK, Italy, Spain, China and Japan will account for two-thirds of the global market and for almost 60 percent of the growth in spending.

11. Gli Anticorpi monoclonali: - Cosa sono - Come si fanno - Dove si usano - Quanto costano

12. Come si usano nella ricerca? Immuno- fluorescence Flow cytometry Immunoblotting

13. Come si usano nella ricerca? Immuno- precipitation Immuno- histochemistry Purification ELISA

14. Biotechnology & Therapeutics

15. Immune Responses 1. Humoral: B lymphocytes produce antibodies recognizing an antigen from foreign substance. Antibodies are then secreted into blood stream. 2. Cell-mediated: Mature T lymphocytes - antigen responding, response control, and response mediating cells

16. Glossary Antibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte). Antigen (antibody generating substance) is any agent, such as a chemical or microorganism that is recognized by the antibody. Not all antigens are immunogens (e.ghapten). Immunogen : Any substance to which an animal responds by making antibodies. All immunogens are antigens. Epitope : the part of a target to which an antibody binds, also known as an antigenic determinant Antigen binding site - relatively small region of an antibody that binds to the antigen.

17. Immunogenicity Ability of a molecule to induce an immune response Proteins, peptides, carbohydrates, nucleic acids, lipids Must be larger than 3000-5000 daltons - if not Carrier protein e.g. BSA Thyroglobulin Antigen (hapten) Carbodiimide Glutaraldehyde MBS–Heterobifunctional reagents

18. Glossary Antibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte). Antigen (antibody generating substance) is any agent, such as a chemical or microorganism that is recognized by the antibody. Not all antigens are immunogens (e.ghapten). Immunogen : Any substance to which an animal responds by making antibodies. All immunogens are antigens. Epitope : the part of a target to which an antibody binds, also known as an antigenic determinant Antigen binding site - relatively small region of an antibody that binds to the antigen.

19. B A Antigen C D What are epitopes? Different portions of a target that can be recognized by different antibodies Each individual antibody only recognizes a single epitope Polyclonal antibodies recognize many different epitopes

20. Antibody Structure Mike Clark

21. Struttura anticorpale 2 CATENE PESANTI Un dominio variabile VH Tre domini costanti CH 1-2-3 2 CATENE LEGGERE Un dominio variabile VL Un dominio costante CL

22. How VH genes are made in vivo 51 VH genes 27 D genes 6 J genes CH1 Rearranged V gene VDJ CH1 CH2 CH3 mRNA After somatic mutation

23. How are antibody genes rearranged in vivo? The VH chain is made up of the combination of V, D and J segments, the VL Chain is made up of V and J only. There is no D segment in VL genes.

24. Variability plot: human VH

25. 22 98 VH S S 31 35 49 66 101 110 1 117 FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3 (4-30) Rearranged V gene structure

26. Antibody Production Polyclonal: Antibodies are collected from sera of exposed animal, - or - a combination of monoclonal colonies is combined. Can be any animal: Rabbit, Goat, Horse, Rat, Sheep, etc… Suite of antibodies recognizing multiple antigenic sites of injected biochemical.

27. B C A Antigen D B C A D Production of Polyclonal Antisera Lymphocytes a b c d Antibody b Antibody a Antibody c Antibody d

28. Immunizations Animals: Rabbits, mice, goat, donkey Immunogen: Antigen – 50 - 1000g Adjuvant – non specific stimulator of immune response Route of Injection: sub cutaneous 1st Intra-dermal, intra-venous, intra-peritoneal, Boosts: at 4 weeks intervals - can rest the animals Test bleeds: to determine response in terms of specific antibody Further boosters Collect blood POLYCLONAL Antiserum

29. Freund's adjuvant • is a solution of antigenemulsified in mineral oiland used as an immunopotentiator (booster). • The complete form, Freund's Complete Adjuvant,(CFA or FCA) is composed of inactivated and dried mycobacteria (usually M. tuberculosis); • the incomplete form (IFA or FIA) lacks the mycobacterial components (hence just the water in oil emulsion). • It is named after Jules T. Freund.

30. Freund’s Adjuvant is used as a water-in-oil emulsion. It is prepared from non-metabolizable oils (paraffin oil and mannidemonooleate). If it also contains killed Mycobacterium tuberculosis it is known as Complete Freund’s Adjuvant. Without the bacteria it is Incomplete Freund’s Adjuvant. Freund’s Adjuvant is designed to provide continuous release of antigens necessary for stimulating a strong, persistent immune response. The mycobacteria in Complete Freund’s attracts macrophages and other cells to the injection site which enhances the immune response. For this reason, the Complete Freund’s Adjuvant is used for the initial injections.

31. Advantages of Polyclonal Antisera B C A D Antigen B C A D Lymphocytes • Antiserum recognizes many different epitopes on the target • Can usually be used for many different research procedures • Immunohistochemistry, immunofluorescence, immunoprecipitation, ELISA, precipitation assays • Can be affinity purified to eliminate the non-specific binding antibodies b a c d Antibody b Antibody c Antibody a Antibody d

32. Disadvantages of Polyclonal Antisera • Antiserum is composed of a mixture of high and low affinity antibody populations • Antiserum is composed of a mixture of antibodies with different specificicities - not all recognize the target of interest • If the animal has had an infection, antibodies against the infecting organism will be present - can lead to non-specific binding • Quantity of antiserum is limited by amount of serum and life of immunised animal. • Immunogen must be pure.

33. A Perspective of Monoclonal Antibodies • What are monoclonal antibodies? • What are the principles involved in their production? • How do they differ from conventional antisera?

34. & Georges Köhler (1946-1995) Cesar Milstein (1927-2002) Anticorpi monoclonali -1975 Premio Nobel - 1984

35. Cesar Milstein Georges Kohler Isoelectric focusing immunoglobulins made in hybridoma cells

36. Definition: • Mono: One • Clone: A strain of cells descended form a single cell • Antibody: A molecule of animal origin that has immunolgical activity only against the antigen to which it was made

37. Definition of A Monoclonal Antibody • Mabs comprise identical antibody molecules. • They only recognise a single antigenic determinant. • They are secreted by a clone of hybrid cells in culture.

38. ARGOMENTO: PRODUZIONE DI ANTICORPI MONOCLONALI Definizione Uno degliesempipiu' significatividell'induzione e selezione di mutanti di cellule somatiche e dellafusionecellulare e' datodallaproduzione diibridomi. Gliibridomisonodegliibridi di cellule somaticheottenutimediantefusionetralinfocitie cellule di unalineaderivata da cellule tumorali di mielomamultiplo. Lo scopo e' quello di immortalizzareilinfociti in modo da poterprodurre in vitro, per tempi indefiniti, e quindi in grandiquantita', glianticorpicheilinfocitinormalmenteproducono e secernononelcorso del lorostadiodifferenziativo finale di plasmacellula.

39. How to 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

40. Ag Outline Of Monoclonal Antibody Production 2) LYMPHOCYTES (Lymphocytes secreting AB are preferentially selected for fusion) FUSION 1) MYELOMA CELLS Confer immortality on lymphocytes HYBRID CELLS 3) CLONE To separate cells 1 cell divides to give clone of identical cells 4) CELLS 1 type AB MAB1 MAB4

42. How to 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

43. Production of Monoclonal Antibodies Immunisation with antigen Ab titre from serum Ab1 Ab2 Abn

44. IMMUNIZE THE MICE

45. IMMUNIZE THE MICE

46. Immunize the mice - Inoculation • The mice are aseptically inoculated with the antigen combined with an adjuvant. • Normal dose per mouse is between • 50 and 100 micrograms of protein. • Inoculations are performed every 21 to 28 days. Inoculations are done either sub-cutaneously or intra-peritoneally.

48. https://www.jove.com/video/2771/manual-restraint-common-compound-administration-routes-micehttps://www.jove.com/video/2771/manual-restraint-common-compound-administration-routes-mice

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