Immunisation techniques. Antibody (polyclonal and monoclonal) production, purification, labelling. Hybridoma technology, fermentation Department of Immunology and Biotechnology University of Pécs
Structure of an antibody e p y t io d I Isotype
Antibody and antigenic determinant
Antibody and antigenic determinant (epitope)
Types of antigenic determinants
A complex antigen generates polyclonal immune response
Monoclonal antibodies: antibodies with the same specificity, produced by a cell line derived from only one activated B cell, able to recognise only one epitpe Y Y Polyclonal antibodies: mixture of antibodies with different specificities, product of more B cell clones, recognise different epitopes of an antigen Ag Y Y Y Y Ag
Immunization Selection of the adequate animal: Monoclonal antibodies mouse or rat Policlonal antibodies rabbit, sheep, goat Antigen: hapten (5-10 kd >) carrier native or modified molecules, cells Administration: intramuscular, subcutaneous or intracutan
Immunization
Immunization Adjuvants: delay the absorption of the antigen and nonspecifically stimulate the immune system (activation of macrophages) resulting in increased antibody secretion. Examples: Incomplet adjuvants: contain mineral oils (incomplete Freund) Complete Freund: heat-inactivated Mycobacterium tuberculosis suspended in mineral oil Aluminium-hydroxid gel Muramyl-dipeptide (Mycobacterium tbc.) Bacterial vaccines (e.g. Bordetella pertussis) co-administration with immunogens can increase the antibody production against these immunogens Liposome
Purification of polyclonal antibodies Antibodies are extracted from the sera of immunized animals. IgM precipitation of Ig fraction with chemical methods: based on molecular weight and solubility euglobulin precipitation Further purification with gel chromatography Density gradient centrifugation
Purification of polyclonal antibodies IgG: Ion-exchange chromatography Affinity chromatography (Staph. aureus protein A, Streptococcus protein G)
Purification of polyclonal antibodies IgA, D, E: multiple step methods IgA: after Zn precipitation; ion-exchange chromatography and gel filtration IgE: affinity chromatography Immunosorbent methods: Antigen specific isolation on the surface of Sepharose, cellulose or polyacrylamid gels
Antibody testing Testing of titer and specificity should be done in the same system where the antibody will be applied e.g.: ELISA flow cytometry immunohistochemistry
Hybridoma technique, production of monoclonal antibody With the hybridoma technique we are able to select a specifically activated (e.g. antibody production), but limited life span immune cell and keep it in vitro (proliferation). The basis of the technique is that we fuse in vitro an activated immune cell with a B-cell/plasma cellderived tumor cell line.
Myeloma Plasma cell Immortality Antibody production Hybridoma Immortality and antibody production
Hybridoma production
Hybridoma production 1. Cell fusion: spleen cell (from immunized animal) + non-secreting mouse myeloma (Sp-2/0-Ag14) + PEG 2. Selection: Selection of the desired hybrides (spleen cell + myeloma) Sp-2 cells are deficient for HGPRT and TK enzymes (purine and pyrimidine synthesis), but spleen cells have them, so by the use of a selective medium (HAT) we can select the spleen cell Sp2 hybrids Pathways of DNA synthesis
Selection of the antibody producing hybrid cultures, testing (ELISA) Hybridoma production Cloning: dilution of the antibody producing cells so that on a 96 well plate statistically less than 1 cell is seeded in a well. Testing antibody production of monoclones. Antibody produced by a single clone of cells: MONOCLONAL ANTIBODY Monoclone
Hybridoma production 4. Mass antibody production: (1) production of hybridoma supernatant in vitro; (2) administration of hybridoma cells ip. in mice (ascites); (3) fermentor (artificial mouse).
Applications of monoclonal antibodies Preparative methods Protein purification Analytical methods (Diagnostic purposes, research) Identification and isolation of lymphocyte subgroups and clones Tumor detection and imaging Routine laboratory techniques- serology Therapy Tumor Autoimmune diseases Chronic inflammation 1g gold ~ 20 1g mab ~ 800 If you have the monoclone
Fluroescent dye e.g.: FITC Enzyme e.g.: HRPO Biotin Conjugation
Enzyme conjugates Enzyme converts substrates that manifests in colour reaction. HRPO, ALP Immunohistochemical slides
Fluorescent dye conjugates FITC FITC FITC, PE PE Imunofluorescence Flow cytometry
Toxin conjugates A toxin conjugated to an antibody binding to the surface antigen of the target cell will be internalized by the target cell. Then the toxin is released into the cytoplasm and exerts toxic effect (e.g. Ricin can block the protein synthesis of the cell). Ricin
Radioisotope conjugates γ ray emission, low dose Diagnostic purposes radioactive imagining Diagnosis of tumors and micrometastases α, β ray emission, high dose Treatment of tumors
Bispecific antibodies TUMOR Effector cell NK Tc Macrophage A bispecific antibody is created by chemical cross binding of the Fab fragments of two different Igs. Tumor specific Fab part Effector cell specific Fab part: CD3 Tc CD64 (FcγR1) high affinity FcγR; Mφ CD89 FcαR; Mφ, granulocytes
Therapeutic monoclonal antibodies registered by FDA Name Specificity Type Application Year Orthoclone OKT3 CD3 Murine Graft rejection 1986 ReoPro GpIIb/gpIIa Chimeric Cardiovascular diseases 1994 Rituxan (mabthera) CD20 Chimeric Non-Hodgkin lymphoma 1997 Zenapax CD25 Humanised Graft rejection 1997 Remicade TNFα Chimeric Crohn s disease, rheumatoid arthritis 1998 Simulect CD25 Chimeric Graft rejection 1998 Synagis RSV Humanised Respiratoric synctitium virus infection 1998 Herceptin Her-2 Humanised Metastatic breast carcinoma 1998 Mylotarg CD33 Humanised Acute Myelogenous Leukemia (AML) 2000 Campath CD52 Humanised B cell chronic lymphoid leukemia (B-CLL) 2001 Zevalin CD20 Murine B cell non-hodgkin-lymphoma 2002 Erbitux EGFR Chimeic Colorectal carcinoma (EGFR+ tumors?) 2004 Avastin VEGF Humanised Colorectal carcinoma 2004 Tysabri α4β1/7 integrin Humanised Multiple sclerosis 2004
Murine antibodies The first therapeutic monoclonal antibody registered by FDA. OKT3 mouse monoclonal anti-cd3 (1986). This therapy is quite effective but its repeated use is accompanied with severe immunological side effects: HAMA (human anti-mouse-antibodies) The constant part of an Ig is conserved, but there are some differences between human and mouse Igs. HAMA can be detected after 8-12 days of treatment, the peak concentration is after 25-30 days.
Chimeric antibodies The Fv region of the chosen monoclonal antibody gene is fused to Fc part of a human Ig gene. Approximately 75 of a chimera Ig % is of human origin. The specificity of the antibody is similar to that of the original mouse antibody. The in vivo half life and effector functions of the Ig are similar to those of the original human antibody. HACA (human anti-chimeric-antibodies) Less immunogeneicity, but sometimes HACA can be detected.
Humanised antibodies The genes of the CDR regions of a mouse monoclonal antibody are implanted into the genes of human antibody. More than 90% of the antibody is of human origin. The specificity of the humanized antibody is similar to that of the original mouse antibody. The in vivo half life and effector functions of the humanized antibody are similar to those of the original human antibody.
US Food and Drug Administration nomenclature of therapeutic antibodies Chimeric mab Fully human mab Murine mab Humanised mab mab xi zu li tu ci u o Monoclonal antibody Chimeric antibody Humanised antibody Immunomodulatory antibody Antitumor antibody Cardiovascular antibody Human Murine The Lancet Volume 369, Issue 9573, 12 May 2007-18 May 2007, Pages 1641-1657