4/5 - Antibodies Flashcards
What does the abbreviation “Ig” mean?
IMMUNOGLOBULINS
also known as Antibodies
proteins made by B-cells
to recognize a foreign substance
What does the abbreviation “Ag” mean?
& define
ANTIGEN
Any material or molecule capable of REACTING with an ANTIBODY
to form a AB-AG complex
does NOT have to be IMMUNOGENIC
Immunogen
= substance capable of inducing immune response
What is a Fab?
List one molecular functions in immunology.
Antigen Binding“ARMS”
BOTH Light + Heavy Chains
What is an Fc?
List two of its molecular functions in immunology.
Fc Receptor Binding“STEM”
ONLY Heavy Chain
What are the antibody H and L chains?
How are they held together?
2 Heavy Chains
Identical 50kDA
2 Light Chains
Identical 25 kDA
DISULFIDE LINKAGES
@ the hinge region
What is the variable (V) region of the antibody?
Contains BOTH
Heavy & Light Chain
The TIPS of the Fab Regions where there is
ANTIGEN BINDING
has specificity to a antigen
What is the antibody constant (C) domain?
Also contains Both Heavy & Light Chain
but also Both Fab & Fc
Does not have variability, does not bind the antigen
But is where BIOLOGICAL ACTIVITY is
Determines the DIFFERENT CLASSES OF AB’s
What are the lambda and kappa chains?
The TWO TYPES of LIGHT CHAINS
subclasses of AB’s differ in Kappa or Lambda Chains
and in:
that can match up with any of the types of heavy chains
5 different heavy chains
Describe the general features shared by all classes of antibodies.
SHARED FEATURES
Same overall structure, Fab / Fc / Hinge Region
SUBCLASSES differ in:
Heavy chain sequence
&
Kappa or Lamda Chain
What are the biological functions of this antibody?
IgG
Predominant AB in Blood / Lymph
2x Heavy // 2x Light chains
- *Glycosylation** (carbohydrate modification)
- regarding biosimilars, this is where we can make changes that influence binding / immunogenecity*
PASSIVE IMMUNIZATION by xfer of AB’s
due to its persistance / high half life
- *AGGLUTINATION**
- *agglutinate** insoluble antigens like bacteria/virus
- -> precipitate soluble multivalent antigens
OPSONIZATION
Activation of COMPLEMENT
Fc region used to stimulate NK CELLS in cell-mediated cytotoxicity
can NEUTRALIZE TOXINS
such as tetanus / botulinus / snake venoms
What are the biological functions of this antibody?
IgA
Major AB in External Secretions:
saliva / mucus / sweat / gastric fluid / tears
Dimeric Secretory form
has 2 extra peptides, + joining J chain
- *Monomeric Serum form**
- *2 alpha-H-chains** + 2Kappa or 2lamda
Main defene against LOCAL INFECTIONS
due to its presence in secretions
- *Agglutinating AB**
- *multivalent** –> good for defense against viruses
does NOT activate complement system
since it is SECRETED, complement is in blood/lymph
What are the biological functions of this antibody?
IgM
5 subunits = Pentameric
2H + 2L along with additional Disulfide bonds & J-chain
- *Valence of 5**
- insted of 10, there is steric interference*
carbohydrate mods on heavy chains
AGGLUTINATION
very efficient due to 5 seperate antigen binding = polyvalency
helps with phagocytosis
ISOHEMAGGLUTININS
naturally occuring AB’s against RBC antigens, ABO bloodtypes
COMPLEMENT
ony a single IgM molecule is needed
–> extracellular killing of pathogen
What are the biological functions of this antibody?
IgD
secreted WITH IgM
surface of B-cells, where it is
co-expressed w/ IgM through alternative splicing
Monomeric
Secreted –> small% of serum abs
has 1 less C domain vs IgE
unknown functions
thought to be involved with elimination of B-cells
that are expressing self-reactive ABs
What are the biological functions of this antibody?
IgE
similar to IgD, but with an EXTRA C DOMAIN
Monomeric
Parasitic Infections
- *HYPERSENSITIVITY // ALLERGIC Reactions**
- *Fc Region** –> binds w/ high affinity to MAST CELLS & BASOPHILS
Crosslinking
IgE binds to Fab Regions –> stimulated cells to degranulate
& release histamine / heparin
–> also release arachidonic acid
What are the VH (heavy chain) and VL (light chain) domains?
How do the VH and VL chains contribute to antigen binding?
VH & VL are what make up the VARIABLE Region
This region is how Antigens are recognized through:
2 Seperate Polypeptides
Quaternary Structure
Describe the kinetics of immune system synthesis of antibodies in the primary and secondary responses to antigen.
Relate the kinetics of immune system synthesis of antibodies to important therapeutic aspects of immunization.
- *3 Phases**
- *Exponential -> Steady -> Declining**
IgM comes FIRST , then IgG
After the SECOND immunogenic stimulus –> FASTER & HIGHER response
10 days vs 3 days
Describe the process of AFFINITY MATURATION, and relate this process to the efficacy of vaccinations.
After recombination, antigen stimulation causes CLONAL EXPANSION
which triggers processes that change the
CDRs of stimulated B-Cells
(complementary determining regions)
ex = Somatic Hypermutation
Describe the process of isotype switch recombination,
and relate this process to the efficacy of vaccinations
- *KEEP the AG specificity** but
- CHANGE THE FUNCTION* / different AB class
**S(switch) Region** is present on the **Heavy** **C-Region** genes is what mediates the **class switching**
Cytokines from T-cells –> B-cells DNA synthesize switch
Describe the process of V(D)J joining, and relate this process to B-cell maturation and the efficacy of vaccinations
VL (variable Light) region
is coded for by two seperate gene segments = V & J
V(D)J Recombinase Complex
is what performs the gene rearrengements
- *Kappa (K) Light Chain Gene Rearrangement**
- *40** different Vk genes & 5 Different Jk Genes = 200 combinations
- Lambda (l*) Light Chain Gene Rearrangement
- *40** different Vl genes & 4 Different Jl Genes = 160 combinations
D comes with the heavy chain, is not always present
Overview of Ig Production
VDJC = forms the Heavy Chain
VJC = forms the Light Chain
Alternative Splicing
will produce BOTH IgD &IgM
Describe how antibody affinity can increase through somatic hypermutation
type of Antigen-Griven Affinity Maturation
Point Mutation in the CDR
Initial Binding –> stimulates proliferation of B-cells that make AB
Secondary binding –> SOMATIC HYPERMUTATION
will SELECT for AB’s that bind more TIGHTLY to the pathogen
VVVV
variant that is BETTER THAN THE ORIGINAL
will help enlarge AB = variety / specificity / B-cell response
What is Activation Induced Cytidine Deaminase?
activation-induced cytidine deaminase(AID), i
s involved in class switch recombination,
somatic hypermutation, and gene conversion, the
three major pathways that generate diversity of
antibodies.
Describe how alternative splicing allows
- *IgM and IgD** on the same B cell to have
- different heavy chains* but the same antigen-binding specificity.
Immunoglobulin Class Switch Recombination
The CH region changes
which results in a different heavy chain
but the VH region stays the SAME
entire Light chain (lambda + Kappa) stays the same
Describe how B cells can switch from making IgM to making IgG, IgE, or IgA
Ig Class Switching** = **Isotype Switching
- *B-Cell** binds the AG –> takes it in & processes it
- *AG** parts bind to MHC2 molecules –> move to cell surface
- *present’s** the processed AG –> T-Cells
B-cell is stimulated to switch production = IgM / IgD –> IgG/A/E
CYTOKINES from the t-cells influence the isotype
- *SAME VARIABLE REGION** = same antigen specificity
- but different BIOLOGICAL ACTIVITIES*
Describe the actions and effects of V(D)J recombinase.
aka RAG1/2
Performs the GENE REARRANGEMENTS
for the:
Kappa + Lamda Light Chain Gene Rearrangements
&
Heavy Chain Gene Rearrengements
same process & enzyme are used in T-cells for rearrangement of TCR (T-cell receptors)
Describe the process of producing polyclonal antibodies
Peptide Design –> Carrier Protein
+
Primer Design –> E.Coli Expresssion + purification
VVVV
Immunization of LIVE ANIMAL (rabbit / mouse / goat)
VVVV
Blood Collection
AB Purification
Describe the process of producing monoclonal antibodies
- -Isolate the SPLENOCYTES (HGPRT POSITIVE) from the dead mouse
- -Mix with “Immortal” cell line (MYELOMA CELLS)
- -That are HGPRT NEGATIVE
- -Mix with “Immortal” cell line (MYELOMA CELLS)
-
-Together with HAT Medium
- -Only the FUSED w/ HGPRT-CELLS can grow
- -Unfused ones will DIE, because they are NOT immortal AND they do NOT have the HGPRT + (selected by the HAT MEDIUM)
- -Collect and analytical tests for ANTIBODIES
- -Chosen hybridoma is grown to PRODUCE THE DESIRED MONOCLONAL ANTIBODIES
Compare and contrast monoclonal and polyclonal antibodies in terms of:
1) time to produce,
2) relative cost to produce,
3) relative potential for non-specific recognition
4) relative availability over a long period of time (many years).
Polyclonal AB’s
Cheap & Fast, limited to the blood supply of host
100s-1000s of ABs each with different affinity & epitope specificity but the Same antigen
more likely to exhibit CROSS REACTIVITY
Monoclonal AB’s
Expensive & Timely, but are INDEFINITE PRODUCTION
completely reproducible = same mAB
AB’s from each clone recognize just 1 EPITOPE
Describe the challenges associated with producing Biosimilars of therapeutic mAbs.
a. Will they recognize the same epitope?
b. Can they trigger a different physiological response as compared to the original mAb?
c. Why, or why not
Recognize the SAME ISOTOPE, but
Subtle changes in the chemistry of the mAB may induce large functional changes
Glycosylation - 1 way in which AB’s can introduce variation
sugars / sugar branching can affect the biological activity
As a practicing pharmacist, will you be able to advise use of therapeutic mAbs and their biosimilars interchangeably?
What will you need to know before you might do so?
Somewhat, Yes.
The MANUFACTURER & The Manufacturing process
Small production fluctuations, such as those related
to cell culture pH, temperature, and media ingredients,
also may impact the final product,
introducing micro-heterogeneities such as
alternative disulfide pairings/ disulfide shuffling, deamidation, (methionine) oxidation, crystallization of N- terminal glutamine residues, and partial enzymatic cleavage
Heavy Chain Gene Rearrangements
V(D)J Joining
The Variable Region is constructed from 3 Gene Segments
VH / DH / JH
with MANY Combinations
unlike LIGHT chains, there are Multiple Genes for the C-Region
D & J Segments code for the
CDR = complementarity determining region = hypervariable region
which directly contacts w/ antigen
V(D)J Recombinase complex
is the same for all VDJ gene rearrangements
Describe how immunoglobulin rearrangements might give rise to lymphomas.
Errors in HEAVY-CHAIN Gene Rearrangements
can move the MYC ONCOGENE
to the IgH locus –> INCREASING MYC EXPRESSION
tells the cells to divide & proliferate –>
LEUKEMIA / CANCER
B-cells are making MYC instead of Antibodies
IgG Subclasses
1-4
IgG1
most abundant –> protein antigens
- *IgG2**
- *bacterial capsular polysaccharide**
IgG3
activator of pro-inflammatory responses, by trigerring the classical complement pathway
- *IgG4**
- least abundant*, often generated following repeated exposure to the same AG or during persistant infections
