13: Antibody drugs Flashcards
what are polyclonal antibodies
Aby secreted by many different clones of B cells against different antigenic epitopes
polyclonal antisera is generated by
inoculation of a suitable animal host species with multivalent antigent
what are phylogenetic considerations
considering the relationship between the donor Ag species and the recipient Aby producing species
when producing polyclonal antibodies, the more distant the species the _____ the immune response, and the ____ Aby is generated when exposed to donor Ag
greater
more
_________________ animals may be required to minimize infectious disease cross over to recipient of Aby
specified pathogen free animals
describe how polyclonal antibodies are made
disease Ag presented to animal
animal produces Aby from lots of different B cell clones
different types of Aby for this one Ag now put back in infected human
in goat anti-mouse IgG, what is the host species and the antigen presenting species
host = goat
antigen presenting = mouse
in horse tetanus antitoxin, what is the host species, what does it neutralize
horse = host
neutralizes tetanus toxin
what 2 kinds of cells do you need to make hybridomas
immortal myeloma cells + mouse splenocytes
which cells are deficient for enzymes that recycle nucleotides for DNA synthesis in hybridomas
myelomas
how many times on average are mouse immunized
3
how to screen that the hybridoma is secreting the appropriate Aby
ELISA
flow through cytometry
western blotting
list the 4 steps of hybridoma production
- immunization
- fusion
- screening
- subcloning
bacteriophages are
viral molecules that infect bacteria
phage display technology generates Aby like fragments called
single chain fraction variable (scFv)
describe the process of phage display
coat antigen on plate
add phage library
wash and see which sticks- repeat 3x
infect e.coli with it to amplify + rescreen or ELISA
ELISA to find antigen binding clones
IPTG induced large scale expression of identified clone
what antibody is found in egg yolks
IgY
how to make your desired antibody in an egg
immunize chicken with antigen 2x at ~21 weeks with pathogens, toxins, biomarkers, allergens, etc and it’ll lay an egg for you with the specific IgY
applications of chicken egg Aby
diagnostic reagents
biomedical applications
how to get biotech products from natural sources
extraction + protein separation and fractionation
describe recombinant techniques for biotech products
recombination of DNA genes + vector genes which are then introduced to host cells to secrete the coded proteins
what are recombinant techniques used for
to manufacture what kind of drugs/ ligands?
to manufacture larger protein ligands and cytokine drugs
describe chemical synthesis for biotech products
amino acids attached step by step to a solid support
- there is a solution phase and a solid phase
peptides = \_\_ AA proteins = \_\_\_ AA
peptides = 2-40AA proteins = >40AA
what are semisynthetic approaches to making biotech products
chemical mod of natural peptides
issues with absorption of peptide drugs
poor absorption across membranes + low oral bioavailability
peptide drugs typically have a ___ Vd and ___ half life
low, low
peptide drugs are prone to ______ and/or ______degredation
aggregation
chemical degradation
why do peptide drugs have low Vd
large size
molecules larger than __ can’t pass through glomerular membrane for urinary excretion
70kDa
protein _____ _____ increases when GFR decreases = greater diffusion across barrier
sieving coefficient
chemical degradation breaks ____ bonds resulting in
covalent
chemical structural changes
physical biotech drug structure stability depends on
protein secondary, tertiary, or quaternary noncovalent interactions
which degredation breaks noncovalent bonds, chemical or physical
physical
oxidation is ____ degradation, aggregation is _____ degradation
chemical
physical
denaturation is the loss of ___ or ___ structure, resulting in irreversible or reversible changes
secondary or tertiary
what sterilization methods should not be used on proteins
heat, gas, ionizing radiation
biologics should be assembled under ___ conditions and a final _________ should be done
aseptic
final filtration sterilization (pre filter, then 0.22um filter)
T or F: excipients used for biologics can not be sterilized by heat either
F- equipment and excipients sterilized separately from protein API, then combined
pyrogen removal mainly targets
endotoxin (LPS) from gram - bacteria
how are pyrogens removed
ion exchange chromatography
reverse osmosis generated water for injection
dialysis, charcoal, oxidation, dry heating
what are some excipients used in biotech drugs for parenteral formulations
solubility enhancers
anti-adsorption/ aggregation agents
buffers
what is the shelf life of aqueous biologics
<2 years
stability of aq biologics depend on
pH, ionic strength, temperature, excipient stabilizers
why do we freeze dry biologics
because the presence of water promotes physiochemical degradation = can extend shelf life to >2 years without it
what excipients does freeze drying require
bulking agents
collapse temperature modifier
lyoprotectant
dried, compact biologic tablets are mostly used for ______ and are applied ______ through compressed air powered rifles
used for animals by vets
applied subdermally
what kind of oral biologic formulations seem to show improved GI uptake
lipidized
what are some limitations to IM and SC
may result in metabolic inactivation before hitting systemic circ
bioavailability limited by circulation to specific area
variable residence time and disposition
T or F: the transdermal route avoids first pass
T
what is the primary challenge of the transdermal route
proteins and peptides do not penetrate the stratum corneum well
how can we increase ability of drugs to penetrate transdermally
penetration enhancers- microneedle patches, sonophoresis, iontophoresis
what is a noninvasive approach that avoids first pass
nasal administration
what is a barrier for bioavailability in nasal admin
large molecular weighr
why is buccal sometimes preferred over GIT
less protease and acid degradation
what is a problem with pulmonary admin route
lung irritation and induction of immune response
what are biotech products
when living organisms are involved in the production or modification of a product
what are biologic response modulators
immunomodulators that target disease causing mechanism
what are biosimilars
a biological product that is highly similar to a reference product and show no clinically meaningful difference from reference product in terms of safety, purity, and potency
how do biologic drugs differ from small molecule drugs
structural complexity
manufacturing techniques
what are some patient related factors that can influence biologic ADRs
genetic predisposition to produce neutralizing antibodies
expression of the endogenous eq of the product
comorbid conditions
T or F: immunogenisity from biologics are usually mild
F- range from irrelevant to lethal- unpredictable
excipients are added in order to increase biotech drug ____________
stability and preservation
maintain tonicity
facilitate drug delivery
avoid immunogenicity
protein based therapeutics may cause an ____ response leading to AEs, or drug ____ upon repeated exposure
immunogenic
drug clearance
excipient categories include
pH modifier tonicity agents bulking agents wetting and//or solubilizing agent antioxidants antimicrobial preservative chelating and/ or complexing agents
on average, how many excipients does a biological drug product contain?
4.45
T or F: small molecule oral medications contain less excipients on average compared to biological drug products
F- bio = 4.45, molecule = 8.8
what are the 4 most common excipients in biotech drugs
water
sodium chloride
polysorbate 80
sucrose
how can clinicians provide further investigational evidence of ADR to medications
skin prick testing + other diagnostics
describe BRM therapy
a type of treatment that mobilizes the body’s immune system to fight cancer
describe the cytokine scavenging Aby drug in terms of bone resorption
binds cytokines so that it can’t release signals and stimulate osteoclasts to breakdown bone
what is chimeric mAb
an antibody that has components from both mouse and human
what is the prefix associated with mouse Aby
xi
how to make humanized mAb
graft CDRs from a mouse antibody onto a human variable region framework
what prefix is used for humanized Aby
zuman
fully human Aby prefix
umab
fully human antibodies have been achieved using ____ + _______
phage display and mammalian cell culture
infliximab is a __ aby and binds and neutralizes ___
mouse chimeric Aby
binds TNF-alpha
adalimumab is a ____ aby that binds _
fully human antibody
TNFalpha
what are drug delivery systems
specially designed formulations or devices that can deliver the drug cargo exactly where it needs to be at a controlled rate + minimize systemic exposure
list some reasons why DDS are needed
increases ease of administration
increase deposition at specific site = decreased AE and lower dose needed
resolve formulation issues
T or F: companies may get FDA approval for a drug tablet, they can then make this drug into a solution and sell it
F- only what form is approved can be made
what are the 5 features of an ideal DDS
ease of administration able to control the release of API able to deliver API at site needed low immunogenic potential/ hypersensitivity nontoxic
what are some DDS delivery vehicles
liposomes, polymers, nanoparticles
what are the 6 factors affecting selection of route of administration
drug characteristics ease of administration mechanism of action onset of action + duration quantity of drug to be administered
T or F: DDS can alter the physiochemical and PK properties of API
T
what is naked antibody targeting
unmodified antibodies used as BRMs, cytokine scavengers, receptor agonists/ antagonists, etc
what is rituximab target? is it mono or polyclonal
monoclonal Aby that targets CD20+ B cells in RA
antibody drug conjugates are
antibodies with targets that deliver drug cargo
how to make antibody- drug conjugates
must use immunoconjugation strategies- only a small number of protein FG serve as practical targets for bioconjugation
what protein FG can be targets for bioconjugation to add drugs onto Aby
primary amines
carboxyls
sulfhydryls
carbonyls
what are theranostics in Aby
antibody isotope conjugates for imaging and therapy
fusion proteins created through joining of 2 or more genes that originally coded for separate proteins describes
antibody fusion protein constructs
how do antibody fusion protein constructs work
newly bound antibody and protein can act as decoy receptors for inflammatory proteins = bind and don’t activate complement to decrease inflammation
abatacept uses __________ technology
antibody- fusion protein
how are immunoliposomes made
vortex lipids with ethanol = forms unilamellar liposomes by extrusion through stacked polycarbonate membranes
post insertional technique to transfer targeting peptide into bilayer of liposome
at what temperature are immunoliposomes made
stir at 37C for 5hrs, then 19h at room temp
liposome release can be triggered by
pH decrease external alternating magnetic field hyperthermia light ultrasound
what are quadromas
bispecific/ bifunctional Aby for drug delivery targeting
what are quadromas useful for
crosslinking 2 distinct targets
rapid immunodiagnostic kits
what is the current chemical fusogen of choice for hybridomas
PEG
T or F: immunization of mice to produce hybridomas require adjuvants
T
viable hybridomas are ___ medium selected
HAT- a medium without functional thymidine analogues = only cells with active TK enzymes can survive
do peptides and proteins penetrate the stratum corneum of the skin
no
which of the following may result in metabolic inactivation before reaching systemic circulation + are limited by circulation to specific area
- IV
- IM
- SC
- Nasal
- buccal
2, 3
which show greater heterogeneity, biologic drug products or small molecule drugs
biologic- protein folding + bonds
which antibody therapy is more likely to have to be injected IV, dufliximab or yuzumumab?
dufliximab because it’s a mouse chimeric Aby = more likely to be recognized by immune system than yuzumumab a human antibody = must be injected IV instead of SQ
