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Monoclonal Antibodies
- Antibodies (Abs). Also known as immunoglobulins (Ig).
- Comprised of 2 heavy chains and 2 light chains
- Monoclonal Abs bind specifically to a single site (epitope) on a particular antigen
- Abs are produced by B lymphocytes
- Because of their specificity and ease of generation, they are extensively used as therapeutics (“passive immunotherapy”) and as diagnostic and research tools
-They can be generated in large (unlimited) amounts in vitro
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B cells develop in the bone marrow hematopoietic stem cells and lymphoid stem cells T-cells and B-cells
progenitor = pro-B cell (B220+)
precursor = pre-B cells: heavy-chain rearranged
immature B cell: IgM + light-chain rearranged
mature B cell: IgM + IgD + an antigen encounter in spleen or lymph nodes; goes to periphery
Terminally differentiated cell = plasma cell, periphery, Ig secretor
Antibodies are made by B-cells
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Domain structure of an immunoglobulin molecule
} Fc
}FabFragment ,antigen binding
Fragment , crystallizable
disulfidebonds
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Laboratory fragmentation of antibodies
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ComplementaritydeterminingRegion = “CDR”
Hypervariableregion
Ig molecule showing polarity, disulfides, carbohydrate
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Opsonization
Complement activation
Antibody-dependent cell-mediated cytotoxicity (ADCC)
Transcytosis
Fc functions
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Opsonization: Direct uptake of bacteria coated with antibody molecules
Complement activation: Activated complement proteins lyse cells by making holes in their mebranes(e.g. bacteria)
Antibody-dependent cell-mediated cytotoxicity (ADCC):Killer T-cells use antibodies on their surface to target cells with an antigen and kill them.
Transcytosis:Antibody-antigen complexes are taken up (endocytosed) on on side of an epithelial cell and directed to theother side, where they are exocytosed
Fc functions
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Multigene organization of Ig genes
–light chains: V, J (variable) and C (constant)–heavy chain: V, D, J, (variable) C (constant)
Mechanism of Ab gene rearrangement
Recombination signal sequences (RSS)–flank V, D, J gene segments
–V-RSS------RSS-D-RSS---------RSS-J
11IgGkappa gene rearrangement
SPLICING
SPLICING
+ SOMATIC HYPERMUATION
+ SOMATIC HYPERMUATION
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Antibodies can participate in host defense in three main ways
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Got this far
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ADCC = antibody-dependent cell-mediated cytotoxicity
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MAb therapy targets
Inflammation
Autoimmune disease
Graft rejection
Heart disease (thrombosis)
Cancer
Viral infection
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Therapeutic strategies
Mabs straight
Mabs fused to other protein binders (e.g., soluble receptors)
Mabs fused to cytotoxic agents (toxins, radionuclides)
Toxins: ricin (stops protein synthesis)calicheamicin (DNA breaks)
Radionuclides:90Y = yttrium111I = indium
17Problems of mouse MAbs
1) Fc portion limited in its ability to interact with Fc receptors of human cells.
2) Lower serum half-life
3) Development of human anti-mouse antibodies (HAMA)
A) Retreatment results in allergy or anaphylactic shock
B) Retreatment is less effective
Solutions via recombinant DNA genetic engineering :
1) Chimeric mouse-human antibodies: Hu V-region fused to mouse C regions
2) Humanized mouse antibodies, Parts of V-region from human interspersed with mouse CDR V-regions
3) Human antibodies (fully), via transgenic mice carrying human immunoglobulin genes(Medarex, Abgenix, Kirin)
Breedveld, Lancet 2000 355:9205
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Sponsor company Generic name US trade name
mAb type Therapeutic category
US approval
Johnson & Johnson Muromonab-CD3 Orthoclone OKT3
Murine Immunological
‡ 19.06.1986
Centocor Abciximab ReoPro Chimeric Hemostasis 22.12.1994
Biogen IDEC Rituximab Rituxan Chimeric Antineoplastic 26.11.1997
Protein Design Daclizumab Labs |
Zenapax Humanized Immunological 10.12.1997
Novartis Basiliximab Simulect Chimeric Immunological 12.05.1998
MedImmune Palivizumab Synagis Humanized Anti-infective 19.06.1998
Centocor Infliximab Remicade Chimeric Immunological 24.08.1998
Genentech Trastuzumab Herceptin Humanized Antineoplastic 25.09.1998
Wyeth Gemtuzumab ozogamicin
Mylotarg Humanized Antineoplastic 17.05.2000
Millennium/ILEX
Alemtuzumab Campath Humanized Antineoplastic 07.05.2001
Biogen IDEC Ibritumomab tiuxetan
Zevalin Murine Antineoplastic 19.02.2002
Abbott Adalimumab Humira Human Immunological 31.12.2002
Genentech Omalizumab Xolair Humanized Immunological 20.06.2003
Corixa Tositumomab-I131 BEXXAR Murine Antineoplastic 27.06.2003
Genentech Efalizumab Raptiva Humanized Immunological 27.10.2003
Imclone Systems
Cetuximab Erbitux Chimeric Antineoplastic 12.02.2004
Genentech Bevacizumab Avastin Humanized Antineoplastic 26.02.2004
MAbs approved for human therapy
EGF-R colon cancer
HER-2/neu (EGF2) breast cancer
CD33 leukemia (AML)
VEGF colon cancer
IgE asthma
Respiratory infectionSynciitial Virus
IL-2 immunosupressant
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Monoclonal antibody generation
- Cells needed myeloma cells, mouse spleen cells - antigen administration Kohler and Milstein - hybridoma formation via cell fusion- selection mutants required: defer
- antibody generation cDNA cloning- engineered MAbs expression vectors- refinement chimeric, humanized, human (defer)
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Monoclonal antibodies via cell hybridization
Selects forrare hybrid cells
21Reducedmyeloma hybrid
Isoelectric focusingimmunoglobulinsmade inhybridoma cells
Georges Kohler
Cesar Milstein
Unreducedmyeloma hybrid
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Mab Fusion Proteins
Other protein-binding proteins: natural receptors in soluble form
Analogous to MAbs and make use of the Fc portion of the antibody molecule:
Example: Enbrel (etanercept):Anti-rheumatoid arthritis drug Soluble TNF receptor fused to the Fc IgG1 domain (TNF= tumor necrosis factor)
Ties up TNF, blocking its inflammatory functionFc domain dimerizes the receptor, which increases its affinity for TNF.Fc domain increases the half-life of the protein in the bloodstreamAmgen + Wyeth
Still experimental: anti HIV drug PRO 542 Soluble CD4 (HIV receptor) fused to IgG2.
Tetrameric (4 V-regions replaced)Reduced Fc function (since IgG2 < IgG1), Better half-lifeProgenics
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Single chain antibodies (scFv)
Ag binding site
15 AA linker
Phage display selection of scFvSource of sequence:PCR from genome or mRNA, add randomization