ANTICORPI MONOCLONALI

x Antigen Spleen cells Immunization Cell fusion Monoclonal antibodies Adapted from Milstein (1980) Scientific American, Oct. p.58 1 2 3 4 Antigen m 1 2 3 4 Spleen cells Immunization + Myeloma B cell 1 2 3 Cell fusion 1 2 3 4 4 x 1 2 3 4 1 2 3 4 Monoclonal antibodies Antiseum Pure single Ab A mixture of all Ab If a single B cell was picked up and cultured, then it will produce only one kind of antibody. But B cell can not survive well in the culture. Each B cell produces only one kind of antibody, which binds to its specific antigen. Conventional antiserum is the mixture of all antibodies produced by B cells from spleen. After immunization, the mouse spleen contains B cells producing specific antibodies. If B cell is fused with myeloma, the fused cell might be cultured and produce antibody. Each hybridoma line can produce pure single antibody, called monoclonal antibody. Myeloma cell can be cultured in the test tube, but can not produce useful antibody.

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

Hybridoma 1975 Kohler and Milstein Multiple myeloma (human disease) Fusions generated by Sendai virus or PEG The last important break-through was by Kohler and Milstein in 1975. They took an antibody producing but mortal B-cell and fused it with an immortal myeloma cell producing a single antibody chain. The result was a hybridoma cell that was immortal and produced one type of antibody. This opened the way to unlimited production of antibodies of a single type and specificity. These antibodies have found application in diagnosis, purification and therapy. Bence Jones proteins in urine http://www.immunecentral.com/images/immune_series/immune29.gif

Creation of monoclonal antibodies (mAb’s) 1. Hyperimmunize mouse with Ag 2. Fuse B cells with tumor fusion partner (+ PEG) 3. Limiting dilution (96 wells) to fractionate fused cells in HAT media selection HAT media (hypoxanthine, aminopterin, thymidine). B cells die (mortal, HGPRT-positive) Tumor cells die (HGPRT deficient and cannot utilise salvage pathway) Fusions live (HAT Resistant and immortal)

Hybridoma selection (HAT media) The de novo pathway can be inhibited using aminopterin, which inhibits the transfer of methyl groups from activated dihydrofolic acid. cells need Hypoxanthine and Thymine as sources of purines and pyrimidines for the salvage pathway. Myeloma cells are HGPRT- and cannot create nucleotides in the salvage pathway. Plasma cells are HGPRT+ and can utilize hypoxanthine in the salvage pathway.

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

Affinity Purification How to purify your MAb Affinity Purification • Or use Ag or epitope to make affinity column Monospecific antibodies http://www.tulane.edu/~wiser/methods/handouts/pwpt/17_mab.pdf

IMPIEGHI DEGLI ANTICORPI MONOCLONALI Protein purification Identification and isolation of cell sub-populations using fluorescence cell sorting. Tumor detection and imaging Tumor killing Diagnostic reagents. Drug Detoxification Catalytic antibodies

IMMUNODIFFUSIONE

Immunoprecipitation At appropriate ratio of antigen:antibody, complex forms an insoluble aggregate. Technique can be used to purify specific protein from a mixture Co-immunoprecipitation – interacting proteins also found in the immunoprecipitate

Impieghi dell’immunoprecipitazione Determinazione delle concentrazioni relative di anticorpi o di antigeni Confrontare diversi antigeni tra loro Stabilire la purezza di un preparato antigenico

Immunodiffusione Quando l'immunoprecipitazione avviene all'interno di un gel si parla più propriamente di immunodiffusione. Immunodiffusione radiale Immunodiffusione doppia immunoelettroforesi

Diagrammi di possibili Modelli di precipitazione con la doppia immuno- diffusione eseguita su un antisiero e 2 diversi preparati antigenici A: epitopi identici B:nessun epitopo in comune C:identità parziale

Separazione elettroforetica degli Ag Archi di precipitazione IMMUNOELETTROFORESI Separazione elettroforetica degli Ag Archi di precipitazione

Determinare se un paziente produce quantità troppo basse di Ab isotipi, come si verifica in alcune immunodeficienze Si può vedere se produce se produce un eccesso di alcune proteine, quali albumina, immunoglobuline

Immunoelettroforesi quantitativa Ponendo in grafico l’altezza degli archi contro la concentrazione Si otterrà una retta di taratura che può servire per determinare la concentrazione di Ag in un campione incognito

(Presenza o assenza di gonadotropina) TEST DI GRAVIDANZA: ES. DI INIBIZIONE DI AGGLUTINAZIONE (Presenza o assenza di gonadotropina)

IMMUNOFLUORESCENZA

Gli Ab sono marcati con molecole fluorescenti

Le molecole fluorescenti assorbono luce a una determinata lunghezza d’onda (eccitazione)e la emettono ad un’altra lunghezza d’onda (emissione). Se gli Ab sono marcati con i fluorocromi, possono essere rilevati attraverso la valutazione della luce colorata emessa in seguito all’eccitazione a una determinata lunghezza d’onda La luce emessa può essere rivelata con un microscopio a fluorescenza I coloranti possono essere coniugati alla regione Fc di una molecola senza modificarne la specificità Coloranti: fluoresceina, rodamina, ficoeritrina

Immunofluorescense Very good rapid diagnostics IFA of Ehrlichia chaffeensis in DH82 cells, 400X Bullous pemphgoid with linear deposits of IgG at dermal-epidermal junction Very good rapid diagnostics

Citofluorimetria e fluorescenza Queste tecniche si basano sull’impiego di Ab fluorescenti a hanno carattere qualitativo FACS (fluorescence-activated cell sorter) Quando una cellula passa davanti al raggio laser, la luce viene deviata dal rilevatore e questa interruzione del segnale laser viene registrata. Le cellule che possiedono l’Ab fluorescente legato ai loro Ag di membrana vengono eccitate dal laser ed emettono luce, che viene registrato da un secondo sistema, localizzato ad angolo retto rispetto al raggio laser. Lo strumento conta ogni cellule quando passa davanti al raggio laser e registra i livelli di fluorescenza emessa dalla cellula Un computer collegato crea i diagrammi con il numero delle cellule in ordinata e la loro intensità di fluorescenza in ascissa

SEPARAZIONE DI CELLULE MARCATE CON Ab

DIAGNOSTICA

ELISA – Enzyme-Linked Immunosorbent Assay Test antiserum Test antigen

Visualization of reaction

ELISA to test MAb production itself The dark blue spot represents a positive clone. The light blue spots are positive controls, the next two wells to the right are negative controls www.ch.tum.de/wasser/weller/ formerprojects_im...

SAGGIO ELISPOT

Saggio radio-immunologico Il metodo è basato sulla competizione tra un antigene non marcato e una quantità fissa dello stesso antigene marcato, per il legame con un numero limitante e costante di siti anticorpali. Ab + Ag* Ab/Ag* Ab/Ag* + Ag Ab/Ag Ab/Ag* E' necessario costruire prima di tutto una curva di taratura, % conc. Ag Marcato legato a Ab Tratto lineare Conc. Ag non marcato

FRAZIONI DI Ab

The Fab fragment contains the antigen binding site Could be cleaved out by papain digest; pepsin digestion even better, as it produces F(ab’)2 Often it is enough for diagnostic purposes – and it is easier to produce Anti Botulinum toxin Equine FAB fragment product made by DoD at time of Gulf War, anti-ABCDEFG Hinge region www-structure.llnl.gov/ fab/fab_tutl.htm

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

IMPIEGHI COME FARMACI

About 25-30% of women who have metastatic breast cancer overexpress HER-2 (EGF) receptor Determination of HER2-protein overexpression (semiquantitative DAKO Hercep Test™) Fluorescence in situ hybridisation (FISH) of HER2 amplification www.roche.com/pages/ facets/9/herc.htm

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

Problemi con la terapia basata su mAb Gli Ab di topo sono visti come estranei dal sistema immunitario umano Innesco della risposta immunitaria contro gli HAMA (human anti-mouse antibody) Danni nell’ospite mAb causerebbero meno problemi (volontari cercasi??!!) Soluzione: anticorpi ibridi uomo-topo

Anticorpi chimerici: Combinazione della regione variabile di topo con la regione costante di uomo Anticorpi umanizzati: Ab combina solo gli aa della regione ipervariabile di topo con il resto di Ab umano

as a recombinant protein in the protein expression system Anticorpi umanizzati 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

Topi transgenici con segmenti genici di anticorpi umani

Xenomouse (topo con IgH e Igk umani) Abgenix http://www.sh.lsuhsc.edu/ intragrad/micro/micro297/4

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

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