Antibodies and Gene Therapy Flashcards
Monoclonal Abs mAbs
Polyclonal Abs
3 reasons not why
What do you need though?
What is the problem?
Even if a B cell produces what you want?
What do we need?
Middle Ear catheter
Hows it work?
Is it invasive?
Examples of ELISA tests
Compatibility of Protein drugs with other delivery devices (PUMPS)
- Reports of adsorption of protein to tubing in IV infusion: 3 things Drugs that were effected
- What could the compatibility affect? 2 things
- Considerations should be followed just like?
3
Protein Composition
What type of chain and what is it made up out of?
How many naturally occuring types?
What are 4 ways their chemical makeup varies?
Comparison of Genome Editing Tools
3 on the slide
Genome editing
How to cut DNA Precisely at a desired location
One of this is pretty common and uses a metal
Examples of Variables to Consider during IV admin of Protein Therapeutic DRugs
What I just said but examples
Example 1 continued
Protein folding
Driving force based on what sequence?
4 points? Vs. What other point?
- Collapse?
- What interactions?
- What bonding?
- This list is not?
Vs.
- Protein and water? Entropy?
Humoral Immunity and Antibody
Critical for protection against? Or for?
How to most vaccines elicite protection?
Antibodies in Human healthcare
How many approved Ab drus?
How many in clinical trials?
How many approved vaccines?
Antibody half life extension
This is talking about how protection is transfered to a baby.
IgG binds to?
How? what is the mechanism?
Other otic diseases?
5 different ones
Which one can be given steroids and antioxidance prophylaxtically
Sudden Sensoroneural Hearing loss
Goals for Formulation
The protein needs to be stable how do you do that? 2
What factors do you need to consider with parenteral forms? 4 things
Why are delivery devices important? and why? what type of things can happen? 2 reasons
Catheters- Cochlear Implant
What have they developed in recent years?
To prevent rejection of the wires the coat them with glucocorticoids that slowly release to reduce inflammation and build up of scar tissue
Generating human Abs
3 ways to accomplish this
- Humanization- Loop sequence from mouse tranplanted to human Ab and doesnt lose affinity- Less immunogenic, Really want to make completely human Abs
- Human Abs from transgenic (humanized) mice- These have a human immune system, But sometimes they cant make Abs with high enough affinity for the disease
- Generation of Human Abs from Combinatorial libraries in vitro 2 kinds can be make Phage display technique, Isolation of spleen cells PCR amp, Ab genes are then exprssed in bacteria the 2 libraries- From healthy unimmunized people, Biased libraries made from immunized individuals There are also synthetic libraries
Model of Multi state process of Non-Native Protein aggregation
What is the best and most effective form of the protein?
Because of this what do you want to prevent?
The goal is to keep the protein in as mich of the monomer form as possible or to have it readily be reversed back into the monomer so the drug can still be active
Anti Tumor Vaccines
Long t1/2 application Agglutination test
Fancy word for?
Protein Folding
We say that it cannot be a? What paradox?
What about the timescale of protein folding?
Therefore? 3 points
- What type of folding and what type of intermediates?
- What do solvent interactions do?
- What type of proteins in vivo
Formulation stresses affecting protein aggregation
Role of Formulation variables 3 things
- Different Classes of?
- Protein?
- Multi-use?
Role of interfaces 3 things with examples
- Liquid to
- Liquid to
- Liquid to
Role of Processing 3 things with examples
- Operations?
- Keeping something still?
- When you are giving it
Ear anatomy the middle and inner ear
and zoomed in on the Cochlea
Potential Gene and Cell Therapy Targets
What are the 4 types of targets?
Microwick
What does it do?
And how does it work?
Is it invasive?
Drug Labels: Selected sections that comment on Storage and Stability
- Simponi must be?
- Do not use beyond?
- Do not?
- Do not?
- Simponi is provided in?
- Simponi doesnt contain?
- When should you not use the solution?
TALENs?
Transcription Activator-like effector nuclease
Hybridoma tech 7
Reversible self association of mAbs at high protein concentration
What are the interactings between the mAb monomers that allow them to be reversible?
3 one is obvious
Monoclonal Abs uses in biotech and medicine
12 things?
Drug examples 2
B cell Hybridomas Secrete monoclonal Antibodies
Polyclonal B cell problem
Fuse B cells?
Bad picture of middle ear
Mechanism of Protein Stabilization by Certain Solvent additives
When sucrose is preferntially excluded from the surface of proteins what happens?
Which type of protein has more surface area?
According to Le Chats principles?
What do proteins do to overcome thermodynamically unfavorable conditions?
Modeling Inner ear Drug delivery Concentration gradients and graphs
Bottom is distance
Stating with cochlea
RWM Structure
- Outer
- What type of cells?
- Rich in what two things?
- Continuous with?
- Junctions?
- Middle
- When is elastic density highest?
- What 3 things are in this?
- Inner
- What type of layer epithelial shape with what type of extensions?
Transposase Mediated Gene Delivery
Protein stabilization using Excipients
Chemical class 7
MOA 6
Examples of post-translational modifications and degradation of proteind in vivo
7 things
Examples of commercial products high protein concentration SUBQ delivery 6
Protein Degradation pathways In Vitro
Chemical Pathways 8
Just know the differences between the two
Physical Pathways 4 and 4 subs in last one
Sterile prep of protein drugs and IV Admin
Sterile processing during manufacturing of protein drugs usually involves?
Sterile handling may involve? 2 things
Stability within?
Immuno PCR
What does it do what is it able to detect?
Combines what 2 tests? This provides extremely?
Whats the point to developing High Concentration mAb formulations?
Sub Q admin typically requires?
- mAb range?
- How is the SubQ injection limited?
- How is at home use facilitated?
What are 5 pharmaceutical challenges involved?
- If it will disolve
- If it is too?
- Realistic?
- Stability in?
- _____ related to 2nd point
ELISA
Compatibility of New formulations designed for at home use
2 brand names
One is Lyapholized and one is a solution
What are some issues that at home use can cause for stability
Clinical trade off between Control of Drug level and Risk
picture chart thing
7 different modifications
ELISA Variants
Hybridoma Technology
Whar are the total of 7 things things you need?
- Mouse spleen that is immnized with what you want
- Use agents to facilitate fusion of membranes
- Low success rate here, Myeloma cells dont have HGPRT, these myelomas cannot be able to synthesize Ab molecules are there own
- Unfused cells cant grow because they dont have HGPRT, unfused normal spleen gant grow forever because of life span, Hybridoma can grow because it has the HGPRT needed for the myeloma cell to survive adn the meyloma partner doesnt die
- Find the ones producing Ab
- Isolate single cells from each ab(+) culture and subculture those for specificty
- Each (+) subculture is now a bunch of clones and they are monoclonal
- Now scale up
IV delivery and protein stability
Setup varies in 4 ways
- ___ and ___ rate
- Size of
- __
- __
IV bags 2
Different? and Different types of?
Admin kits 2
Different types of? and Different?
Hearing
Picture of cochlear frequency
People can damage their hearing at different sites and this is specific to these regions
Protein Stability
Which state is more stable? Unfolded or Folded? Numerically how much more stable?
4 points pertaining to this?
- How many Hydrogen bonds?
- What about small proteins?
- What is only marginally stable?
- Unfolding?
Protein stability refers to?
More cochlear anatomy
What is inside the cochlea?
Why is this important for drug delivery?
Introduction
Recombinant proteins made from different cell lines leading to? AS in what are the 3 types of organisms used for protein things
3 cell types used
Proteins are Sensitive what 4 things are they sensitive to? What makes them not work
Formulation Turning molecules into medicine 4 things
- What do you do for clinical admin?
- What do you need to figure out when the pt is taking it?
- How lng can it last?
- Friendly to who?
Transposase Mediated Gene Delivery
Developing a Stabilizing formulation is Essential
Start with formulation considerations
4 things in the middle
Then you end with?
Role of environmental stresses?
Role of solution conditions?
Effect of environment stresses and solution conditions on: what 4 things?
Cochlear crosssection
Monoclonal antibodies picture
What is the difference between Small Drugs and Large biologics in terms of how they are made up
The antibody universe
Humans have the potential to make how many antibodies? Are antibodies common?
Antibody genese in newly formed? Formed where? They are assembles from combinatorial stitching of 3? and 2?
Newly Form B cells Naive Bcells
How many in the blood?
Encode how many antibodies?
Bind molecules with? Are they selective?
Cells displaying antibodies that bind to “self”? When does a Cell become inert?
Passive Serum Transfer
Antibody serum or purified immunoglobulin transferred to another to?
Horse serum is stilled used for?
How can human blood products be used? These are used to provide what type of immunity? and to what type of peopl? Example?
IVIG is manufactured from?
Evolution of Antibody Technology
Dynamic Behavior of Immunoglabulins
What region connects Immunoglubulin fragments? 2 parts of this?
What is between V and C domains?
2 types of motions
Sidechain?
Applications of CRSPR tech
What does a long half life mean for the drug?
Relieves pressure for development in 6 ways
What does this do overall?
Ven Diagram
Preformulation Characterization:
Determine Degradation Pathways and Physiochemical Stability Profile
4 parts
The end thing in the middle is?
Comonly Prescribed Otics
Antibiotics? 3 points
Analgesics 1
Something that losens earwax?
Steroids?
Antifungals?
CRISPR
Cluster Regularly Interspaced Short Palindromic Repeats
Abs structure and recognition?
What does IgG consist of?
WHat are the variable regions?
Treating the Ab with what can cleave it into what 2 parts?
Generating human Abs
2nd way out of three
Substances that cross the RWM
Some are weird whY?
Ferritin, Albumin, latex spheres very big its weird they can get in
Monoclonal antibody structure
2 portions?
Important physical characteristics 5
- How heavy?
- How many AAs?
- Domain?
- Sugar?
- Type of molecule?
Modeling inner ear drug delivery
LADME?
RWM is like?
How does delivery work?
Essentially a very small hole in a narrowing hallway
Infusion hypersensitivity
Rituximab example
What type of Ab is this? ___/___ anti?
What were the 2 reactions involved?
two main causes?
- Rapid targeting of cells for destruction causes rapid immune response
- Immune response to protect therapeutic
Elisa Results
Why do we care about RSA od therapeutics? Big ass chart
Examples of Protein drug Heterogeneity?
3
Question is what makes the proteins vary non hemogenous
Protein Physical Stability and Aggregation
Two general categories:
Two roles
Non- Ionic surfactant minimizing?
What is an example of this?
What is formulation development for protein drugs?
- Drug at the appropriate?
- Excipients to ensure?
- Primary?
- For admin?
- What type of manufacturing process?
- Develop what methods?
- Define what conditions?
Protein stabilization using excipients divided into what two things?
Chemical Class
and MOA
Stability challenges to Develop [high] liquid mAbs in prefilled syringes
2 things
- Proteins in what type of formulation?
- These are usually?
- Solubility limite? mg/ml
- 3 issues
- Compatability of ___ protein formulation with?
- Componenets of?
- Silicone and Tungten
- What type od device?
The RWM as a gateway to the inner ear
- How long has the RWM been observed?
- Why is this an attractive drug target?
TIL: Stand for? What do you need to isolate?
CAR: Stans for? These are engineered? What do they express? What happens when they bind?
In vitro reprogramming of T cells to recognize tumore cells: Isolated T cells are incubated with? These have been fed wiht? What does this stimulate and activate? These are then expanded to?
Transposase Mediated Gene Delivery
What are the two technologies?
how do they work?
Example 1 of mAb RSA
T cell therapies for cancer Tx
What are the 4 protein structure types?
What are those made up of?
How can mAbs eliminate tumors
3 ways
Advantages of Immunoassays?
5 things
- How small of detection? Meaning it is?
- How can it be done? can you do it again?
- What do you need?
- What type of data?
- What about automation?
Genetics and Antibody production
What are the constant and variable regions?
Antigenicity?
Immunogenicity?
All molecules that have property of Immunogenicity also have property of? What isnt true?
Hapten?
What are the 4 factors that affect a substances immunogenicity?
Phage display
What type od technique is this?
3rd way Human Monoclonal
Factors influencing Immunogenicity of Therapeutic Proteins
Picture of a bunch of stuff
Protein Met Oxidation
Protein Trp oxidation
Physical degradation pathways of protein therapeutics
5 things and these cause what 2 things?
What is the solution to the problem?
Stresses Affecting Protein Physical Stability two general categories
Epidemiology of hearing loss
- How common is congenital hearing loss?
- Some studies suggest?
- What percentage is syndromal?
- Nonsyndromal what percentage is from recessive gene and what percentage dominant?
Intratympanic long acting gels how do they work?
Needle of solution in through the tympanic membrane and edge of round window once temp increases within the boudy the solution turns into a gel and sticks to the Round Window and can be absorbed due to time at active site
This is a less invasive fix
Mechanism of Protein stabilization by certain solvent additives
Examples of stabilizing Solvent Additives 3
Common view? Stabilization properties are not due to? 2 other things
Examples of Chemical Degradations pathwzys
Protein?
Chimeric Antigen Receptors (CARS)
WHat are they
What is difficult about them? What reactions happen?
Pros?
What does the constant region do?
What does the variable region do?
Humoral Immunity:
The ensemble of Ab in the serum and in secretions that confer protection against disease
What are vaccine- Antibodies are elicited by? Many of which contribute to? What dont we know?
What are therapeutic Abs? Discovery of? That are responsible for?
Antibodies for commercially useful dianostics
What 4 properties does a detection system need?
What do these mean?
WHy are murine mAbs not ideal for therapeutics?
NonHuman Abs are only used in two special cases?
Factors affecting RWM permeability
3 things
Membrane
Inflammation
What is passing through?
Picture of organ of corti?
With lots of vocab
What are the two types of hair cells?
What is inside the hair cell?
What are the 3 modulators?
3 causes of Organ of Corti Damage?
4 types of therapy?
The hair cell only have one nucleus so it makes sense that they can die quickly
Antigen what does it mean? What are the 5 ones in the slide?
Ethical Questions in Gene therapy?
Are disabilities diseases?
Do they need to be cured or prevented?
Does searching for a cure demean the lives of individuals presently affected by disabilities?
Is somatic gene therapy (treating adult cells of persons known to have the disease) more or less ethical than germline gene therapy (which is done in egg and sperm cells and prevents the trait from being passed on to further generations)?
Preliminary attempts at gene therapy are exorbitantly expensive. Who will have access to these therapies? Who will pay for their use?
Antidrug Antibody detection
Bridging?
Where is it detected?
What is the critical factor in this?
Human Humoral Immunity
Mining abs from?
Ag binding
Loops of?
What makes the Ag Ab binding tigher?
Typically therapeutic Ab binding?
What are the 4 forces responsible for Ab to Ag binding?
Abs that bind to proteins form a binding site that is?
Abs that bind to small molecules?
Compatibility of Protein drugs during IV admin
3 things
- Manufacturers test for compatibility so carefully review dosing and admin instructions to know:
- What to use
- And the need for?
- Need to know what it looks like?
- Need to know?
Gene therapy
What is the promise?
4 problems in therapy
- What is hard to deliver?
- Other issue with delivery?
- What are some effects that can occur? What has fewer of these?
- 2 things about the engineered cells
Regulatory Guidelines
ICH-M4Q
USP797
Address proper reconsitution
Antibody half lfe what has recently been done?
Mutations of Fc variants what does this do? 2 things
Role of conformational stability?
2 points
Role of Colloidal Stability 2 points?
What happens as a consequence of attractive intermolecular forces?
What influences proximity?
Factors affecting RWM transit
What impairs latex spheres to cross?
What type of ferritin can cross and what type cant?
What 4 substances can increase permeability?
Genome Editing
Methods for Precise insertion or deletion of DNA fragments in Eukaryotic Cell s
3 things
Examples of Chemical Degradation Pathways
5 and examples within those
Formulation stresses affecting protein aggregation
3 roles
Role of: Formulation varriables, interfaces, and processing
From Naive B Cells to Antibodies
The entire question is how do Naive B cells go from niave to ones that produce antibodies?
How are bacterial molecules presented to B Naive B cells?
What are activated B cells and what do they do? What does this introduce?
B cells that display higher affinity for the antigen what happens?
Ultimately what happens to B cells that win? What 2 things are they called?
Route of Admin for FDA approved mABs
So what types of routes are most common
IV, Sub Q, other
most to least
The dynamic Immune System: Continuous Decay and?
How much does the human immune system weigh?
How many lymphocytes?
How many antibody molecules?
During this lecture how many?
Protein Aggregation
Gene Therapy Definition
Insertion of a ___ ____ into the cells of a patient thereby ____ an ____ ____, or conferrring?
Systemic approaches to drug delivery
Flow chart
