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

4/5 - Antibodies Flashcards by Karlo Sison

What does the abbreviation “Ig” mean?

IMMUNOGLOBULINS

also known as Antibodies
proteins made by B-cells
to recognize a foreign substance

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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

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What is a Fab?
List one molecular functions in immunology.

Antigen Binding“ARMS”

BOTH Light + Heavy Chains

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What is an Fc?
List two of its molecular functions in immunology.

Fc Receptor Binding“STEM”

ONLY Heavy Chain

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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.

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*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.

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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

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*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

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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

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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

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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?

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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.

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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

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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*** * **-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 P**RODUCE 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 p**roducing 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-heterogeneitie**s such as **alternative disulfide pairings/ disulfide shuffling, deamidation, (methionine) oxidation, crystallization of N- terminal glutamine residues, and partial enzymatic cleavage**

**_Heavy Chain Gene Rearrangements_** ## Footnote **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** = **c**omplementarity **d**etermining **r**egion = **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**

4/5 - Antibodies Flashcards

(33 cards)