Partridge L13-15 Flashcards
What are useful properties of antibodies when being used in research?
- Diverse
- Specific with high affinity
- Domain structure = stable, facilitates protein engineering
- Multivalent = improved binding, cross-linking can be useful
- Effector properties = useful in some techniques, therapeutics
What are epitopes?
Areas recognised by antibodies. linear – adjacent in sequence (non-conformational) or discontinuous – non adjacent (conformational). Most antigens have several epitopes - these may be different or repeated. Antibodies can bind mono-valently to single epitopes on an antigen or multi-valently to repeated epitopes.
What is immunogenicity?
Ability of an antigen to induce an immune response.
What do we have to consider when looking at immunogenicity?
- Foreignness – sequence homology between antigen and equivalent protein in recipient
- Molecular size - <1000 Da CARRIER PROTEINS
- Chemical composition – aromatic groups, charged residues
- Ability to provoke T cell responses CARRIER PROTEINS
- Use of adjuvants – induce inflammation, “Danger signals”
What are polyclonal antibodies?
Antiserum is product of several B cell clones due to a mixture of antibodies specific to different “epitopes.”
Advantages include it being relatively cheap and being robust (may recognise partially denatured/unfolded antigen). Disadvantages include specific for multiple epitopes, need pure antigen to immunise, can be difficult to standardise.
Antiserum specific for Antigen 1 CROSS-REACTS with Antigen 2.
What are monoclonal antibodies?
Specific for a single epitope, derived from single B lymphocytes.
How are monoclonal antibodies made?
Spleen cells producing antibody from mouse immunized with antigen A are mixed with myeloma cells lacking antibody secretion and HGPRT. Mix and fuse cells with PEG. Transfer to HAT medium. Immortal hybridomas proliferate; mortal spleen cells and unfused HGPRT myeloma cells die. Select hybridoma that makes antibody specific for antigen A. Clone selected hybridoma.
What are the advantages and disadvantages of monoclonal antibodies?
Advantages: highly specific, can be standardised, pure antigen not needed for immunisation. Disadvantages: often conformation sensitive, expensive.
What is a major use of monoclonal antibodies?
defining cell surface molecules e.g. human leukocytes → CD (cluster of differentiation) classification system. Can also identify cell types using monoclonal antibodies.
Rodent antibodies induce immune responses in human patients e.g. HAMA (Human Anti Mouse Antibody), “serum sickness.”
What is the difference between antibody chimeras and humanised antibodies?
Antibody chimeras = Splice mouse V region genes to human C region genes. Mouse V regions still immunogenic.
Humanised antibodies = Splice human framework region genes and mouse CDR region genes a.k.a CDR grafting (facilitated by immunoglobulin domain structure). May lose affinity/specificity. Time-consuming.
What are strategies for generating fully human antibodies?
- TRANSGENIC mice expressing human immunoglobulin genes. Mouse antibody genes replaced by human antibody genes (“Xenomouse”). Mice can be immunized to generate human antibodies by conventional monoclonal techniques.
- ANTIBODY GENE LIBRARIES Antibody V genes are cloned from naïve/immune B cells using a suitable vector and used to make a large “library”. The antibodies in the library can be expressed in bacteria or on the surface of bacteriophage. Antibodies against the desired antigen are selected from the library, usually using phage display techniques.
a. Isolate mRNA from antibody producing cells – blood, lymphoid tissue, bone marrow
b. Reverse transcribe mRNA. Amplify Fab or Fv cDNA by PCR
c. Clone and express Fab or Fv cDNA in bacteria/phage -> antibody library. Phagemid vectors – express soluble protein in bacteria or on surface of filamentous phage particles (M13)
d. Screen antibody phage display library vs solid phase antigen “panning” - Single B cell antibodies. Single antigen-specific B cells from patient blood or lymphoid tissues are isolated using e.g. fluorescent antigen and fluorescence activated cell sorting (FACS). Expressed antibody V genes are amplified and cloned. Useful for generating antibodies against emerging pathogens.
What are nano-bodies?
Single domain antibodies. These are chemically very stable and easily expressed in bacteria and yeast. They are humanisation feasible
What are the four formats of monoclonal antibodies used for therapy?
Fully mouse. Chimeric. Humanized. Fully human.
How are antibodies used as magic bullets to target tumour cells?
- Anti-CD52 antibodies (CAMPATH).
a. recognises leukocytes, good activator of complement and ADCC
b. Use in leukaemias, lymphomas.
c. First humanised (IgG1) antibody used in a clinical trial - Anti-CD20 antibodies (Rituximab).
a. recognises B cells, good activator of complement and ADCC
b. use in leukaemias, lymphomas
c. chimeric antibody - Anti-Her2 antibodies (Herceptin)
a. recognise Her2 (receptor tyrosine kinase, expressed at high levels in ~25% breast tumours)
How are antibodies used in cancer therapy?
Modulation of immune responses – non-infectious disease, cancer.
Depletion of leukocytes e.g. antibodies to CD52, CD3, CD4. Organ transplantation, graft versus host disease, autoimmune disease.
Blocking of cytokines, cytokine receptor, and soluble mediators e.g. antibodies to TNF-α, IL-1, IL-6, complement protein C5 (or their receptors). Inflammatory/autoimmune disease, allergy and over-reactive response to some infections.
Immune checkpoint inhibitors e.g. antibodies to CTLA-4, PD-1.
