biopharmaceutical products derived from endocrine and immune system i Flashcards
what is the function of the immune system
protect body from foreign proteins and invading organisms
what are the cellular and humoral components in innate and adaptive immunity
cellular for innate: phagocytes, NK, mast cells, dendritic cells
humoral for innate: cytokines, complement proteins
cellular for adaptive: T and B cells
humoral for adaptive: cytokines, Ab
what is cell mediated response
by cytotoxic T cells
what is humoral response
Ab produced by plasma cells bind to specific epitopes expressed by pathogen -> Fc domain of Ab bind to Fc receptor expressed by effector cells -> activated effector cells engulf or lyse pathogen -> pathogen clearance
what is the antibody structure
2 heavy and 2 light chains, Fab and Fc domain, Vh Vl, Ch Cl in Fab domain
what is the function of Fab domain
with Ag specificity for binding to Ag
what is the function of Fc domain
to trigger immune response by binding to Fc receptor of effector cells/ complement proteins
what type of post translational modification does Fc domain undergo
glycosylation
what does different AA sequence in Vh and Vl chains allow for
Ag binding specificity diversity
how many CDRs does each heavy and light Fab arm have
3
how many paratopes does an Ab have
2
differentiate between epitope and paratope
epitope is on Ag, paratope is on Fab region of Ab
how many CDRs does one paratope of an Ab have
6 (three from heavy, three from light)
what is antigen affinity
measure of strength of interaction betwen Ab and paratope
what is antigen specificity
measure of goodness of fit between paratope and epitope
what does antigen specificity indicate
indicates the ability of paratope to distinguish similar and dissimilar antigens
what can low antigen specificity result in
cross reactivity where paratope interacts with more than one epitope
what is meant by avidity
strength with which an Ab binds to its target, measure of total binding strength when there is multiple antigenic sites
where are T cell receptors found
on surface of T cells
what are the components of a TCR
an alpha and beta chain, each with a variable and constant region and a transmembrane region and short cytoplasmic tail
what are the components of each alpha and beta chain
each alpha and beta chain consists of two extracellular domains that is glycosylated with carbohydrates
what is the purpose of transmembrane region
to cut across the highly lipophilic plasma membrane
what is the characteristic of the transmembrane region in order to meet its purpose
AA sequence composes of hydrophobic/ non polar to form stable interactions with phosphobilipid layer
what is the drawback of the short cytoplasmic tail
may be too short for signal transduction for T cell activation upon Ag binding
what does the short cytoplasmic tail of a TCR encourage
formation of TCR complex
what is the purpose of the variable and constant region of a TCR
variable region is for Ag binding, constant region contains Cys residues to form disulfide bonds between alpha and beta chains
how are TCR complexes formed
through CD3 dimers
how many CD3 dimers are required to form a TCR complex
3
what are the CD3 dimers required to form a TCR complex
CD3epsilon-gamma, CD3epsilon-delta, CD3zeta-zeta
why are extracellular domains of cell surface receptors important
it allows ligands to bind and trigger changes within receptors so that intracellular components are able to trigger a series of signal transduction reactions which leads to receptor activation and downstream signalling events
how many ITAMs does a TCR complex have and where are they located
each CD3gamma, CD3epsilon, CD3delta have one ITAM
each CD3zeta has three ITAMs
total 10 ITAMs
what occurs to the ITAMs when there is TCR-Ag binding
ITAMs get phosphorylated which is a post translational modification which leads to a series of downstream T cell signalling events hence causing T cell activation
where are B cells developed
bone marrow
how are B cells and its Ab developed
progenitor B cells (original) have heavy and light chain that are able to undergo rearrangement -> rearrangement of IgG -> clones of immature B cells that have B cell antigen receptor -> leaves bone marrow and enter circulation and peripheral lymphoid tissues -> if B cell encounter pathogenic Ag, it will become activated -> produce first line immune response IgM -> gene rearrangement of Fc domain of IgM -> undergo class switching produce IgG instead of IgM -> continuous rearrangement of genes in Fab arms -> produce different hypervariable CDR -> IgG Ab of varying Ag specificity
when is IgM produced
exposure to pathogenic Ag
when is IgG produced
gene rearrangement and class switching
where are T cells developed
bone marrow
where do T cells go too after developing in bone marrow
to primary lymphoid tissue (thymus) then to secondary lymphoid tissue (lymph nodes and spleen)
why is there TCR diversity
genetic recombination of DNA segments in Valpha and Vbeta regions
how many CDR are there on Valpha region of TCR and how is it created
CDR1alpha, CDR2alpha, CDR3alpha
VJ recombination
how many CDR are there on Vbeta region of TCR and how is it created
CDR1beta, CDR2beta, CDR3beta
VJ recombination followed by VDJ recombination
how are memory T cells produced
during an infection, Ag driven expansion of Ag specific T cells clears off invading Ag, effector T cells die after clearing of Ag, remaining Ag specific T cells differentiate and become memory T cells that will have faster and more potent effect if reexposure to same Ag
where are memory T cells found
lymphoid tissues, bone marrow, specific organs
what are MHC molecules
molecules that are cell surface proteins required for adaptive immune system to function
what are the two characteristics of MHC molecules
polygenic and polymorphic
what is meant by polygenic
made up of multiple genes
what is meant by polymorphic
each MHC made up of different alleles that can vary by up to 20 AA
what is the benefit of MHC being polygenic
being made up of different genes suggests different peptide binding specificities -> present different Ag to T cell -> broader coverage of Ag
what is the benefit of MHC being polymorphic
increases diversity of MHC being expressed in each individual -> broader coverage of Ag
what is the function of MHC
bind to peptide fragment (Ag fragment) and present to T cell to aid the immune system in differentiating self from nonself/ foreign pathogen
what is the process of MHC binding to Ag fragment and presenting to T cell
Ag picked up by APC -> phagocytosis -> forms Ag fragments that get bound to MHC II -> peptide-MHC (pMHC) presents to CD4+ -> activate CD4+ to produce cytokines like IL2 -> activate CD8+ and B cell
what type of immunity does activation of CD8+ lead to
cell mediated immunity
what type of immunity does activation of B cell lead to
humoral immunity through secretion of Ab
do MHC molecules have immune function
no
where are MHC I molecules present in
present in all nucleated cells and platelets
are MHC I molecules present in RBC why
no as RBC has no nucleus
what is the function of MHC I molecule
bind to peptide fragments of endogenous Ag and present to CD8+
what are examples of endogenous Ag
normal self Ag, viral components from a virus infected cell, neoantigens produced by cancer cells
where are MHC II molecules present in
APC and B cells
what is the function of MHC II molecules
bind to peptide fragments of exogenous Ag and present to CD4+
what are exogenous Ag
usually foreign Ag belonging to invading pathogens
what are MHC molecules regulated by
IFNalpha and IFNgamma
what are IFN
cytokines
what are cytokines
substances secreted by immune cells
what happens if levels of IFNs increase
expression of MHC I/II molecules increases
why is IFNalpha produced
as an early response to viral infection
what does IFNalpha do
increases transcription -> increase expression of MHC molecules -> T cell activation
what is IFNalpha useful for
enhancement early phase antiviral response
why is IFNgamma produced
immunomodulatory cytokine
what does IFNgamma do
increases expression of MHC molecules -> activate T cells
what is IFNgamma useful for
enhancing immune response
what is the function of a proteasome
protein complex ubiquitously in cells that degrade unwanted or damaged proteins by proteolysis
what is the structure of a proteasome
consists of proteases that cleave peptide bonds so that proteins are degraded into peptide fragments of about 15AA through proteolysis
where are pMHC I formed
in ER/ golgi apparatus
how are pMHC I presented to CD8+
CD8+ has CD8 receptors which binds to MHC I molecule present in pMHC to bring close proximity of pMHC to its TCR on cell surface -> establish binding between TCR and antigenic peptide -> trigger signalling events involving activated TCR
what happens to degraded/ unwanted proteins due to wear and tear from daily cellular activities
unwanted -> undergo proteolysis by proteases into peptide fragments -> picked up by MHC I molecules to form pMHC I -> expressed on nucleated cells and platelets -> present to CD8+ -> CD8 receptor dock to MHC I portion -> bring proximity to TCR -> TCR bind to antigenic peptide -> trigger signalling events
where are pMHC II formed
ER
what happens to exogenous Ag
exogenous Ag taken into APC in endosomes -> undergo proteolysis by proteases into peptide fragments -> picked up by MHC II molecules to form pMHC II -> expressed on surface of APC -> presented to CD4+ -> CD4 receptors dock to MHC II portion -> bring proximity to TCR -> TCR bind to antigenic peptide -> trigger signalling events
what are the classes of cytokines
IFN, IL, hematopoietic growth factors, cytokines, tumor necrosis factors
what is IFN produced by
cells in response to viral infection, tumor, other biological inducers
what is IL produced by
leukocytes
what is TNF produced by
usually present in small amounts but increase production by activated macrophages in response to inflamm conditions
what are the effects of IFN
promote antiviral state in neighbouring cells, regulate immune response
what are the effects of IL
affect growth and differentiation of hematopoeitic and immune cells, regulate immunity and inflamm and hematopoiesis
what are the effects of hematopoietic growth factors
stimulate cellular division and differentiation of blood cells from bone marrow precursors
what are the effects of chemokines
stimulate leukocyte chemotaxis and activation
what are the effects of TNF
pro inflamm and pro apoptosis actions
what is chemotaxis
movement of immune cells in response to chemical stimuli
what are the physical properties of cytokines
small proteins of <20kDa, glycosylated glycoproteins, short half life, secreted out of cells, action short range
what is the generic moa of cytokines
bind to specific cytokine receptor on surface of effector cell to trigger downstream effects
what is the moa of IFN
interfere with viral replication
what is the moa of IL2
immunomodulatory - stimulate growth and differentiation of T cells via autocrine effect, B cells and NK
what is the moa of IL11
stimulate proliferation of hematopoietic cells and induce megakaryote maturation -> incr PLT formation
what is the moa of TNF
hematopoietic effect
what is the biological effects of IFN
induction of cellular resistance to viral attack, regulation of most aspects of immune function, regulation of growth and differentiation
what is the biological effects of IL2
activation of T cell
what is the biological effects of IL11
hematopoietic cofactor
what is the biological effects of TNF
help restore severe deficiency in hematopoeitic cells due to chemotx/ radio tx
what are the kinds of IFN and what are their type classification
type I - IFN alpha, beta
type II - IFN gamma
what type of cytokine is IFNgamma and what does it do
immunomodulatory cytokine which activates resting macrophages and monocytes to increase phagocytic activity (immunostimulation)
what produces IL2 and IL11
T cells produces IL2, fibroblasts and bone marrow stromal cells produces IL11
what are the two types of colony stimulating factors
(grastim) G-CSF, (gramostim) GM-CSF
what are examples of G-CSF
pegfilgrastim, lenograstim
what are examples of GM-CSF
sargramostim
what type of glycosylation in G-CSF and GM-CSF
O linked in G-CSF, N linked in GM-CSF
what is G-CSF useful for
it increases neutrophils so good for treating chemotx induced neutropenia
what is GM-CSF useful for
it increases neutrophils, monocytes, eosinophil counts which is good for accelerating myeloid cell recovery after BMT, antiviral, antifungal therapy, CD
what is antiserum
animal sera containing polyclonal Ab raised by immunising animal with a particular Ag
how is antiserum produced
whole blood collected from immunised animal -> left to clot/ add coagulant -> clotting factors removed -> centrifugate to separate cellular components -> serum obtained as supernatant -> raw serum further purified
how is raw serum further purified
eliminating serum proteins and enriching the fraction of Ig that reacts with target Ag through protein A/G purification or immunoaffinity column chromatography
which method of raw serum purification produces more specific Ig
immunoaffinity purification method
what is the drawback of using antiserum and what is it mainly used for
immunogenicity issues and mostly used for passive immunisation
what are the advantages of monoclonal Abs and what are its uses
highly specific and high homogeneity
highly specific: recombination protein purification
high homogeneity: reproducible for commercial development (target specific therapeutic molecules, test kits, experimental research techniques)
what are the types of mAbs
murine, chimeric, humanised, recombinant human
how are mAbs produced
from one B cell
how are murine mAbs produced
hybridoma technology
what are the limitations of murine mAbs
induces immunogenicity and fail to trigger a number of effector functions and shorter half lifes (30-40h)
what percentage of chimeric mAbs and humanised mAbs are human
~75% and 90%
how are chimeric mAbs produced
replacing AA sequence in Ch and Cl of murine mAbs as not required for binding to Ag, Ag binding Vh and Vl fragments conserved
how are humanised mAbs produced
only hypervariable CDR regions kept
how are recombinant human mAbs produced
using DNA technology to genetically engineer such that AA sequence of heavy and light chains fully human
what are the suffixes for each type of mAbs
murine: -omab-
chimeric: -ximab-
humanised: -zumab-
recombinant: -mumab-
what are some applications that do not require Fc domain
antagonism of enzyme actions, neutralise receptor ligands like hormones and cytokines to counteract overproduction of cytokines, neutralise toxins
what does addition of Fc domain provide
increase half life -> decr freq of administration
what are the types of Ab derivatives
Ig conjugate, F(ab’)2, Fab, ScFv, BiTEs, triomabs
how are F(ab’)2 and Fab derived
cleaved by proteases pepsin and papain respectively
what is the structure of Ig conjugate
radioisotope, cytotoxic cytokine or toxin conjugated to full length Ab
what is the structure of ScFv
AA sequence in Vh and Vl in single polypeptide chain
what is the structure of bispecific Tcell engagers
two distinct Fab regions
what is the structure of triomabs
two distinct Fab regions with Fc region
where does fucose bind to in naturally occurring IgG
Asn297
what is the effect of fucosylation
reduced affinity to subtype of Fc receptor called FCRgammaIII found on effector cells -> less Ab dependent cellular cytotoxicity induction by effector cells
what is the process of TIL
tumor removed from patient -> isolate TILs -> primed and expanded ex vivo -> infused back into patient -> therapeutic TIL reinfiltrate tumor -> recognise and bind to Ag -> attack cancer cells
what are the limitations of TIL
excised tumor mass may have low quantities of TILs
expanded naturally occurring tumor specific T cells are heterogenous with different specificity thus may not be lethal enough
limited or none of expanded T cells have high affinity
what is the process of TCR-T and CAR-T
T cell isolated from peripheral blood of patient -> expand to ensure sufficient quantity -> genetically modified under lab conditions -> develop tumor Ag specific T cells
what makes a TCR Ag specific
Valpha, Vbeta in TCR
how are the T cells cloned
using a retro/lentiviral vector that have high transduction efficiency
what does high transduction efficiency mean
large amount of T cells will carry the desired gene aka high tumor specificity and less heterogeneity
what are the advantages of TCR-T
contain full TCR complex -> can infiltrate tumor
contain full TCR complex with the six CDRs (three on each Valpha and Vbeta) -> allow activity even at low Ag densities
slower onset due to lag time required for TCR to bind to pMHC bc bulkier, but longer duration and extended killing
what are the limitations of TCR-T
since MHC are polymorphic and polygenic, TCR can only recognise and work for patient subpopulation that have same allele
less safe than TIL due to
1. on target off tumor - target normal cell expressing same Ag
2. off target - TCR-T cells not specific and cross react with other antigenic fragments
3. cytokine release syndrome
what is the difference between CAR-T and TCR-T
CAR-T utilises a ScFv as sole extracellular domain so instead of cloning full TCR onto retro/lentiviral vector, clone Vh and Vl of identified Ab onto the vector
what are the advantages of CAR-T
recognise and bind to unprocessed tumor Ag without MHC processing but depends on ScFv specificity and its likelihood of binding to lookalikeness
what are the limitations of CAR-T
only bind to cell surface Ag, ineffective against solid tumors
ScFv may drive CAR-T cells into an Ag independent mechanism
only activated at higher Ag densities and strength of interaction depends on ScFv
faster but weaker killing due to the fact that only one subunit aka ScFv that binds to Ag which leads to weaker CAR signalling and activation
on target off tumor due to B cell aplasia (attacking normal B cell expressing the same target)
even more CRS
compare the difference in structure of gen 1-4 CAR-T and its benefits/ limitaitons
gen 1: ScFv with CD3zeta - still very short cytoplasmic tail, insuff for signalling
gen 2: ScFv with CD3zeta and CD28/4-1BB - second activation allows for stronger signalling
gen 3: ScFv with CD3zeta and CD28 and 4-1BB
gen 4: ScFv with CD3zeta and CD28 and 4-1BB with transgene - transgene activation expresses cytokine like IL12 which exert autocrine and/or paracrine effect on T cells at target site and activate more T cells to eliminate cancer cells
where are checkpoint molecules found
expressed on T cell surface
what happens if ligands bind to checkpoint molecules
suppresses T cell activity
what are examples of checkpoint molecules expressed on T cells
cytotoxic t lymphocyte associated protein (CTLA-4) and programmed death 1 (PD-1)
what does CTLA-4 compete with and bind to
compete with CD28 also expressed on T cell for binding to costimulatory CD80/CD86 on APC
what are the types of cancer vaccines
cell (tumor cell/ dendritic cell)
protein/ peptide
nucleic (DNA/ RNA)
