Notes 20101013 CHEM 731 Meiering Protein Design
From SnOwy - Ed's Wiki Notebook
Some administrative talk for students taking this class for credit... presentations etc., -- I've decided not to take this course for credit -- if I can get something reasonable together for a presentation and submit a paper PDF to Liz by 15:30 Wed. Oct. 20, 2010, I might go for this course as an audit; also include a one-paragraph summary as a word file about why this particular paper is chosen -- send presentations as PPTs by 15:30 Tues. Oct. 26.
Rotamer Libraries
- for modelling and design of proteins
- possible libraries
- parameters in search algorithms
- search algorithms ...
Computational Progress
Title: Progress in computational protein design
- energy functions
- statistical -- or knowledge based methods have improved the most in recent years
- solvent-mediated effects
- design challenges
- we're capable of designing stable globular proteins
- we tend to be less able to predict aggregation
Enzymes
- precise mechanics of enzymes still not well understood
Bound water molecules
- not well modelled in complexes
General notes
- human intervention still needed
- researchers do work on a system they thoroughly understand
Therapeutic proteins
- new area of research
- examples
- monoclonal antibodies
- enzymes
Small molecule drugs
- principle therapeutic agents
- oral bioavailability
- intracellular targets
- ease of manufacturing
- long shelf life (often)
Protein therapeutics
- new, small -- market rapidly growing
- only tools for treating some diseases
- advantage
- high binding specificity
- tunable affinity
- tunable half-life
- disadvantage
- injected -- patient compliance -- not orally available
- extracellular or cell surface targets (due to size)
- expensive production
- short shelf life -- aggregate, degrade
- examples
- immune system in chronic inflammatory diseases
- corticosteroid and cyclosporine A -- binds to many targets related to the immune system
- monoclonal antibody (mAb) -- infliximab -- binds only to harmful tumour necrosis factor α
- immune system in chronic inflammatory diseases
Effective therapeutics
- certain well-validated targets
- finding new targets is expensive
- protein engineering and design is challenging
- protein expression, purification
- drug delivery
- immunogenicity
- some early monoclonal antibodies used to treat tumours triggered immune responses
Design strategy
- improved natural protein
- modified insulin → glargine
- designed monoclonal antibodies
- protein fusions
- appending albumin domain → increase biological half-life
- post-translational modifications
- glycosylation
- pegylation → increase half-life
- engineering -- rational → screening → combination
- rational design
- family shuffling → chimaeric proteins
- random mutagenesis
- directed evolution
- reduced immunogenicity
- glycosylation -- expensive
- pegylation
- epitope elimination -- avoids immune molecules
- humanization of sequences
- use human mAb instead of mouse antibodies
Protein Therapeutics
- mAbs, antibody engineering
- specificity
- engineered with display technologies -- easy
- chimaerization or humanization -- enhances stability (that is, biological half-life not thermal stability)
- can treat cancer and immune disorders
- future: multiantibody cocktails, conjugation to toxic drugs -- a bound target cell is killed by drug
Title: Protein therapeutics -- lessons learned and a view of the future
- small molecules
- ion channel targets
- intracellular enzyme targets
- protein therapeutics
- cell surface targets
- extracellular targets
- enzyme replacement therapy
- challenge -- interference by competitive binding
- large targets
- many receptors for interleukins -- mediate, cause signal cascades
- extracellular receptors used as dummies
- antibodies can be designed as:
- agonists -- binds receptors
- antagonists -- binds extracellular dummies
- Kohler and Milstein -- fused anti-body producing B cells with immortal myeloma cells
- hybridoma
- mAb generation in unlimited amounts
- HAMA -- human anti-mouse antibody
- chimeric, humanized, fully-human antibodies
- antibodies are remarkably stable
- stays together owed to disulfide bonds
- antibodies could be directed at intracellular targets (intrabodies -- new area of research)
- antibody Fc domain (constant domain) can bind to an additional target
Parasite proteins
- Huradin
- protein binding thrombin to prevent thrombosis -- from leach
- Serpin
- pox virus serine protease inhibitor
- prevents artheroclerotic plaque development
Immune response
- anaphylaxis
- we always see a few antibodies to protein therapeutics
- usually not a problem
- depends on genomic background
- purity of protein
- glycosolation state etc., post translational
- aggregation really activates the immune response
- most dangerous if immune response starts to recognize patient's own proteins -- acquired autoimmune syndromes
Title: Strategies and challenges for the next generation of therapeutic antibodies
- target cell displaying antibodies can be used to trigger phagocytosis of target cell
- igG1 antibodies
- clinically validated targets
- first generation 1990's early 2000's
- CD20
- tumour necrosis factor (TNF)
- human epidermal growth factor (HER2)
- epidermal grwth factor receptor (EGFR)
- trends -- require ten years between proof of concept and useful prototype
- second generation
- distinct epitopes -- higher or lower target affinity
- PEGylation
- Fc-fusion
- third generation antibodies
- new epitope targets
- new trigger mechanisms
- better Fc-associated function
- protein modifications to increase homogeneity
- engineering just a Fab stand alone -- the region indicated by a light chain constant + CDR
- antibodies are large -- here are some other scaffolds in trials
- ecallantide -- was approved (Phase 3) then withdrawn this year due to immunigenicity
- adnectin -- 94 aa segment of fibronectin -- FN3 domain
- affibody -- Z domain ...
- DARPin -- ankyrin repeat protein
- many scaffolds under Phase 1 and Phase 2 trials
Title: Therapeutic antibodies for autoimmunity and inflammation
- targets
- autoimmunity
- inflammation
- diseases
- organ transplants
- cardiovascular
- ophthalmological
- display technologies
- increasing specificity of antibody target using phage display
- antibodies can be used for patient diagnosis too
- improve clinical benefits
- cancer drugs can lead to anti-drug resistance -- utilize protein cocktails
- commercial successes lead to competition (mixed blessing)
- current challenges
- shorter half life -- allows elimination after course of action of the antibody
- longer half life -- reduces doses needed
- better delivery and reduced aggregation
- better specificity
- currently not possible to cross the blood brain barrier
- delivery into the eye
Title: High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR
- earlier work
- determinants for residues involved with binding
- altered residues could
- cause aggregation
- improve binding
- got crystal structures
- identified all solvent accessible amino acids
- could simultaneously increase binding in one receptor while decreasing in another
- similar task to alanine scanning for all solvent accessible aa
- affinity always expressed as a ratio with wildtype
- binding affinity results assisted in future design efforts
Title: Engineered antibody Fc variants with enhanced effector function
- "antibody-dependent cell-mediated cytotoxicity" -- the target for modulation
- enhance Fc to FcγR interface affinity
- used a screening approach
- IC50 used to measure cytotoxicity
- results as a ratio against wild type
- number of receptors displayed on surface inversely proportional to efficacy
