Immune-based Techniques in Research and Medicine Flashcards
What properties of antibodies make them useful therapeutic drugs?
Diverse – >108 specificities
Specific, high affinity – KD10-8 – 10-9M
Domain structure – Stable, facilitates protein engineering
Multivalent – Improved binding, cross-linking can be useful
Effector properties – Useful in some techniques, therapeutics
How can we use antibodies to label molecules in complex mixtures?
Fluorescence - Immunofluorescence microscopy
ELISA assay
Radioisotope - Radioimmunoassay and imaging of tumours
Sepharose - Affinity purification
What do antibodies recognise on an antigen?
What are the 2 types of this?
Epitope
Linear epitope - Amino acids adjacent in sequence; Non-conformational
Discontinuous epitope - Non-adjacent amino acids; Conformational
What is immunogenicity?
What factors affect it?
Ability of an antigen to induce an immune response
Foreignness – Sequence homology between antigen and equivalent protein in recipient
Molecular size – < 1kD (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?
Pros and cons?
Mixture of antibodies specific to different epitopes
- Product of several B cell clones
Pros - Relatively cheap, robust (may recognise partially denatured/unfolded antigen)
Cons - Specific for multiple epitopes, need pure antigen to immunise, can be difficult to standardise
What are monoclonal antibodies?
How are they produced? (hint - HGPRT, hybridoma)
Pros and cons?
Specific for a single epitope, derived from single B lymphocytes
Unfused myeloma cells (immortal), lack enzyme HGPRT and antibody secretion
These are fused with spleen cells producing antibody; Fusion induced with PEG
This is a hybridoma; Select the ones that makes antibody specific for your antigen and clone it
Pros - Highly specific, can be standardised, pure antigen not needed for immunisation
Cons - Often conformation sensitive, expensive
Where has the major use of monoclonal antibodies been?
Defining cell surface molecules
Give 2 examples of antibody engineering
Antibody chimeras
- Splice mouse V region genes to human C regions 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 the 2 strategies for generating fully human antibodies? (hint - trans-, libraries)
Transgenic – Mice expressing human immunoglobulin genes
- Mouse antibody genes replaced by human antibody (“Xeno mouse”)
- Mice can be immunised 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”
- Antibodies in the library can be exposed in bacteria or on the surface of bacteriophage
Antibodies against the desired antigen are selected from the library, usually using phage display techniques
How do we get antibodies into gene libraries?
(4 steps)
- Isolate mRNA from antibody producing cells
- Blood, lymphoid tissue, bone marrow - Reverse transcribe mRNA; Amplify Fab or Fv cDNA by PCR
- Clone and express Fab or Fv cDNA in bacteria/phage (phage display) –> Antibody library
- Screen antibody phage display library vs solid phase antigen “panning”
What is a phagemid vector?
Express soluble protein in bacteria or on surface of filamentous phage particles (M13)
How are fully human antibodies selected and generated from Gene libraries?
Antibody V gene libraries made using cDNA from naïve or immune B cells
V genes are fused to a bacteriophage coat protein gene using a phagemid vector
Gene libraries are expressed as V regions on surface of bacteriophage
Bacteriophage expressing desired V regions are selected using antigen binding
Strategies for Generating Fully Human Antibodies include:
1. Transgenic – Mice expressing human immunoglobulin genes
2. Antibody Gene Libraries
What is another one? (hint - B)
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 fully human monoclonal antibodies against emerging pathogens
What are the different formats of monoclonal antibodies used for therapy?
Give a brief description for each
Fully mouse - Antibodies from 100% mice genes
Chimeric - Antibodies with human Constant regions and mice Variable regions
Humanised - Human antibody with some mice genes in variable regions
Fully human - Antibodies from 100% human genes
How do we use antibodies in immunotherapy? e.g. ADCC
What is important for the intended therapy? (hint - specific function needs specific antibody)
Exploit natural antibody binding/effector functions
Important to choose subclass of IgG with appropriate effector functions for intended therapy
- Complement activation - IgG3
- Fc re4ceptors on phagocytes - IgG1 or IgG3
- Fc receptors on NK cells - IgG1 or IgG3
What does mapping of effector site allow us to do?
Precise mapping of effector sites can be used to generate “designer antibodies” for use in therapy
What is glycoengineering and how does it improve antibodies?
Give example
Altering the sugars within antibodies structures can alter and improve interactions
- Removal of fucose improves IgG interaction with FcγRIII (the FcR on NK cells), enhancing ADCC
What can protein- and glycoengineering do for an antibody? (hint half, effector functions)
Antibody engineering to improve half-life (FcRn interaction), improve or remove effector functions e.g. ADCC, complement activation
What can we label antibodies with?
What can we do by labelling antibodies? (hint - tumour)
What can be done with antibody fragments?
Label antibody with drug/prodrug/toxin/radionuclide
Conjugate tumour specific antibody to a drug
Antibody-drug conjugate binds and is internalised, killing tumour cell
For some applications, antibody fragments are useful e.g. better tumour penetration
What is special about specificity of bi-specific antibodies?
What can bi-specific antibodies do?
Bi-specific antibodies form dual specificity links
These can link tumour binding to “effector” cell proteins e.g. tumour antigen + CD3
Decrease in popularity due to side-effects (serum sickness) and discovery of antibiotics meant serum form immunised animals was used less as treatment.
What 2 things might herald a change in this?
Ability to make human monoclonal antibodies
- Slow uptake, but some human monoclonal antibodies are now being used to prevent/treat infection
Conventional antisera human IgG preparations (IVIg) still used in some therapies:
- Neutralise toxins
- Treat infections caused by emerging pathogens
How are antibodies used in therapy? (hint - magic bullets)
Give an example (hint - Her2)
Antibodies as “magic bullets” which target tumour cells
e.g. Anti-Her2 antibodies (Herceptin)
- Recognise Her2 (receptor tyrosine kinase, expressed at high levels in ~25% breast tumours)
Many Cancers Induce Immunosuppression via inhibitory immune checkpoints
How can we use antibodies that block immune checkpoints?
These antibodies have shown beneficial effects in metastatic melanoma and some types of lung cancer
What are some methods of immunosuppression used by cancers?
Produce cytokines which which induce T-Regs
Induce CTLA-4 expression; Has higher avidity for B7 than CD28 so blocks activation
PD-1 (transiently expressed on activated T cells) interaction with ligand PD-L, also induces inhibition
What are CAR-T cells (chimeric antigen receptor)?
How are they made?
T cells “engineered” to recognise a tumour antigen
T cells that are activated with antibodies to CD3 and CD28 are infected with retrovirus encoding anti-CD19 CAR
Infected T cells then express anti-CD19 CAR and then mediate antitumor activity
