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Immunology: Research + Clinic > HC3: Proximity plots, Antibodies, MHC and IgG subclasses > Flashcards

HC3: Proximity plots, Antibodies, MHC and IgG subclasses Flashcards

HC3

1
Q

What does a proximity plot show?

A

Distances for proteins, between its amino acids, show interaction

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2
Q

The proximity plot shows a diagonal as all amino acids are shows on all axes. What do parallel red stripes (red = close) mean to the diagonal?

A

Parallel beta sheet

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3
Q

What do 90 degree extensions / red lines from the diagonal mean?

A

Antiparallel beta sheets

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4
Q

Tight and wide turns

A

Tight turns: the part of close interaction is larger, as the turn is tighter: connected 90d angled line from diagonal and relatively thicker line
Wide turns: 90d angled connected to the diagonal but very small vague red dot line (because in the N > C sequence shown on the axes, the strands are connected so close to each other and the axes, however, the interaction is weaker as the turn is wider)

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5
Q

Alpha helix and beta sheet on proximity plot

A

Alpha helix: thick line (6 AAs), because smaller distance between amino acids in the sequence, larger amount of adjacent amino acids fall under the threshold of the program for being red > wider diagnonal line
Beta sheet: thinner line (3 AAs): larger distance between adjacent amino acids in the sequence.

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6
Q

IgG1 structure HC and LC: how many immunoglobulins?

A

Two heavy chains of 4 immunoglobulins and two light chains of 2 immunoglobulins
Immunoglobulin: one domain of a chain (building blocks, also used for other molecules like immune receptors)

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7
Q

Antigen binding sites of IgG: which chains involved?

A

One HC and one LC (heavy and light chain) per site

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8
Q

Variable regions (immunoglobulins)

A

Immunoglobulins consisting variable loops for antigen binding (N-terminus)

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9
Q

Constant region antibody

A

Fc tail (C-terminus)
> constant immunoglobulins

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10
Q

Hinge region IgG1

A

The CH2 (2nd constant region of one of the HC ) (in Fc part) of one HC crosses with a linker across to the other side to connect to the CH1 (in Fab)
> in this hinge, disulfide bridges are located

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11
Q

What is located on the inside of the CH2 domains of the IgG1?

A

A glycan molecule (carbohydrate)
> normally on outside of molecules
> but on IgG1 on inside of proteins its Fc part
> important for IgG function
> holds CH2 domains a bit apart of each other
> for form and structure
> impacts binding ability indirectly

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12
Q

Covalent interactions in antibody (IgG1)

A

Disulfide bridges
> 2 times HC-LC (at CH1-CL)
> 2 times HC-HC at hinge > hold HCs together (between CH1-CH2)
> Intra-domain for each Ig domain, not needed to hold chains together but for structure as well

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13
Q

Noncovalent interactions of IgG1

A
  • VH-VL
  • CH1-CL
  • CH3-CH3
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14
Q

Types of Ig domains

A

Variable and constant

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15
Q

Structure CL domain and VL domain

A

CL
> seven beta strands in beta sheet
> mirrored point a A-G (N and C termini)
> antiparallel for rest
> D-E-B-A–G-F-C
VL
> D-E-B-A–G-F-C-C’-C’’
Disulfide bond intra domain between B-F
N>C: ABCDEFG

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16
Q

Variable domains (VH/VL) have … beta strands

A

9 beta strands

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17
Q

Where are the three CDRs (variable loops) located for variable domains

A

B-C > CDR1
C’-C” > CDR2
F-G > CDR3

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18
Q

Complementarity Determining Regions

A

CDRs
> three loops, most activity for variable domains: antigen binding

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19
Q

The loops around the beta sheet for constant domains are looped around the beta strands in a complicated manner, why?

A

Stronger interaction
> the structure itself cannot fold open
> Structure itself is linked to itself at many places
> keep everything together
> intrinsic structure is very stable because intradomain interactions (noncovalent)
> cross sheet connections
» same goes for V-type Igs but two extra beta strands

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20
Q

Interdomain noncovalent bonds between C-type Igs (like CH3-CH3 and CH1-CL) and V-type Igs (VH-VL)

A

For C-type: ABED involved
For V-type: C”C’CFG involved

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21
Q

Intra domain disulfide bond at:

A

B-F

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22
Q

Bridge function of antibodies: 3 parts of Abs

A
  • Fab: antigen binding part: CH1-VH and CL-VL
    > the two together: Fab2
  • Fc tail: CH2-CH3 x 2 part > important for Fc receptor binding of C1q binding
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23
Q

How can Fab2 be removed from Fc in lab

A

At hinge region

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24
Q

Ig domains are very compact placed, why is this important?

A

Stable against proteases > because pathogens defend by secreting proteases

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25
Weak spot of antibodies against proteases
Hinge region > cleave antibody into Fc and 2 x Fab to defend against immune response
26
Hypervariable regions HVs
Are the CDRs
27
CDRs
Three loops CDR1/2/3 > six loops, three for VL and VH form one antigen binding site
28
How does variation emerge in CDRs?
- Before antigen exposure: gene rearrangements: diverse repertoire of naive B-cells - After antigen exposure: Somatic Hypermutation (SHM): optimalisation via affinity maturation
29
Variation in T-cell receptors emerge through
Gene rearrangements only: before antigen exposure in LNs
30
Gene rearrangements in HC and LC concept
LC: V and J segment and C segments (for constant part) For each antibody: combination chosen for one V and one J segment while at start many segments available > for HC: V, D and J (and C segments)
31
Which loci are there for an antibody of B-cell
- kappa light chain locus - lambda light chain locus - heavy chain locus > kappa and lambda for different antibodies > specific combination in lambda LC locus and kappa
32
How did VH and VL genes arise?
Through gene duplications
33
Diversity in naive B-cells consists of:
1. combination of diverse V, D, and J segments 2. diversity in joints between VDJ 3. Combination of heavy and light chains (kappa/lambda)
34
Half of the VH segments arisen in evolution are ...
non functional > variation is important for immune system, broad repertoire
35
Which beta strands V segment of HC (VH) / LC (VL)
A-F
36
J segment in VH / VL
Strand G
37
Joint V-J is the ...
CDR3 > for VH, partly coded by D segment > F-G
38
SHM and affinity maturation
- After antigen recognition - Increases after each response (primary, secondary, tertiary) - Selected B-cells > induce mutations in CDRs in GC reactions and select for B-cells which bind antigen best, rest dies by neglect > mutations not in the CDRs: cells neglected because the optimal and compact folding of the Ig domains for structure gets worse and the antigen binding loops do not differ. - Result: affinity maturation
39
High-affinity antibodies only develop when affinity maturation occurs after SHM in GC which is ...
T-cell dependent
40
The SHM process is random and the selection is selective. What is the driver for selection?
Competition for T-cell help > B-cell can bind to antigen better to internalize it (when FDC interaction) and present to T-cell better in GC light zone.
41
Affinities of antibodies, Kd
Kd = ([Ab] * [Ag]) / [AbAg] = koff/kon Affinity high: low Kd, high kon, low koff > Kd determines point of 50% binding of antigen > Low Kd, at a low concentration Ag, the 50% binding is accomplished
42
Avidity
Effective binding strength: multivalent > of an entire antibody
43
Affinity
Intrinsic binding strength: monovalent, of one binding point (CDR)
44
IgM affinity and avidity
- Low affiniy, high avidity, bind multivalent with many points
45
Variation in constant domains established through:
Class-switch > variable parts are held, are antigen specific and are placed on different isotypes
46
Types of antibodies
IgG: very effective IgE: in allergy and immune reaction against parasites: no hinge region IgD: unknown why secreted IgM: primary antibody response, pentavalent with J chain interconnecting IgA: most made, on mucosa ppresent, secretory components and the J chain interconnects the divalent Ab
47
IgM
- Initial response - Low affinity, high avidity - Complement activation
48
IgG
- Extracellular compartments: blood - Important for vaccination - Long half-life (recycling via FcRn, special receptor) - Neutralization, opsonization, complement activation - whole class of immune cells have Fc receptors
49
IgG 1-4 subclasses, why
Different functions > similar structure, different in lengths and hinge
50
IgG subclasses and C1q and FcR binding
- IgG3 best - Then IgG1 - IgG2/4 worse
51
When which IgG subclass?
- IgG1: proteins (and sugars): bacterial and viral infections: always formed, broad repertoire - IgG2: bind sugars (glycan), bacterial infections: shortest hinge, quite rigid Fabs - IgG3: proteins, viral infections - IgG4: proteins (allergens) after chronic stimulation or asymptomatic helminth infections > mostly no infection, less formed, worst in activation of effector functions. From IgG1 to IgG4 response when duration infection: if you do not want too much inflammation but some neutralization long stimulation to prevent damage
52
Big difference in IgG in opsonization and complement activation because:
Difference in the amino acids at the binding sites for C1q and Fc-gamma-receptors and FcRn > FcRn: protein that transfers IgG across placenta to fetus during pregnancy > C1q and Fcy-receptors binding sites at top CH2 towards CH1 > FcRn binding site at CH2-CH3 junction
53
Binding of IgG to C1q and FcR > multiple ways
Monomeric: weak Polymeric: strong: multiple receptors are brought close together > cross-linking FcyR > intracellular signalling induced > cross linking of IgG
54
How does polymeric IgG arise
Opsonization by bringing molecules together and immune complexes: when free antigens in environment: network of IgG interconnected through antigens. > Results in: complement activation and FcR cross-linking (through many Fc tails next to each other: bring receptors together)
55
FcR-IgG avidity measured on a chip
FcR placed on chip > flow of IgG past it > larger complexes lead to higher avidity > FcR can distinguish monomeric and polymeric IgG for binding effectiveness
56
Fab arm exchange
IgG4 can exchange half-molecules in vivo > random process > no large immune complexes > neutralization still > One HC and LC (half Ab) interachanged with half of another > weak disulfide bridges in hinge and noncovalent interactions at CH3 > assymmetric molecules made > low inflammatory antibody
57
Is IgG1 or IgG3 better in complement activation
Depends on the antigen, not always one subclass the best
58
Homology between 4 IgG subclasses
> 90%
59
Allotypes
Genetic variance for constant domains of antibodies
60
Result different allotypes
Subtle structural variations (1/2 amino acids)
61
Effect certain allotypes
Some IgG3 allotypes show enhanced FcRn binding > polymorphisms are important and investigated
62
Allotype variation and Fc-y (gamma) - R binding
The variation affects binding of IgGs to Fc-y-R. > Biosensor chip assay > difference for binding among allotypes for same IgG subclasses
63
IgG glycosylation spots: Fc
1 site per CH2 of the Fc > heterogenous > glycan on the inside of the Fc
64
Variable glycosylation profile of IgG
Different variants of glycans in it > impacts antibody function - In pregnancy (FcRn binding) - Autoimmune disease
65
Most structural variation between IgG subclasses in ...
hinge
66
Fucose
Sugar molecule that may be present on the glycans of IgG > when absent: way better binding (30 x stronger) to Fc-yIII-Ra >> lower Kd
67
IgG glycosylation of Therapeutic antibody to target B-cell lymphoma
anti-CD20 > modified glycan, make it more effective > increased affinity to FcyRIIIa
68
Glycans in variable domain: Fab, which sites
10-15% of IgG it occurs > N-glycosylation sites > NXS or NXT motifs (amino acids) (X is not P) > SHM can induce it by chance! (somatic hypermutation) > mostly in CDRs and the DE loop (the most in DE loop)
69
Glycans in Fab region because
SHM
70
Fab glycosylation effects
- Can contribute to antibody repertoire - Association with several autoimmune diseases - a lot of them without specific difference in antigen binding
71
Fab glycans and antibody stability
May be improved by Fab glycans > can endure higher temperatures without denaturation
72
Therapeutic antibodies: passive immunization
Administer specific antibodies > against pathogens, toxins etc like tetanus toxin > Also against endogenous proteins
73
Antibody therapy: Immune modulating
- Intravenous immunoglobulin (IVIG) > such as ITP > mechanism not well understood
74
Antibody therapy: polyclonal therapies (multiple)
- Non-human anti-toxines (botulism, snake venom) - Intravenous immunoglobulin (IVIG) - Specific human gammaglobulins (anti-tetanus, anti-Sars-CoV-2) - Rhesus D prophylaxis (preventive)
75
Monoclonal antibody therapy: functions and examples
mAbs - Blocking > Anti-IgE: omalizumab > allergy > Anti-TNF: adalimumab > inhibit inflammation - Opsonizing and signalling > Anti-CD20: rituximab > B-cell lymphoma
76
Problem antibodies therapy and solution
Antibody responses against therapeutic antibodies (immunogenicity) > Antibody engineering: humanization of antibodies
77
Types of antibodies with antibody engineering
- Mouse monoclonals from hybridomas (B-cells mouse fused with myelomas > a lot of antibodies for one antigen) - Chimeric antibodies: recombinant - Humanized antibodies: recomninant - Fully human antibodies: phage display and transgenic mice
78
Concept of antibody engineering and humanization is:
Elimination of B-cell and T-cell epitopes
79
T-cell and B-cell epitopes and goal for therapy mAbs
T-cell epitope: linear peptide fragments on MHC B-cell epitope: intact threedimensional protein on surface expressed >> both epitopes should be eliminated on antibodies to stop immunogenicity for therapy
80
Immunogenicity with types of antibodies (not classes, but types as derived from...)
- Mouse: problematic: large part can be recognized > like OKT3 - Chimeric: only variable domains are mouse > like rituximab - Humanized: graft CDRs to human VL, VH > only CDRs are immunogenic > like omalizumab >> limit immunogenicity
81
Human antibodies: phage display, such as in adalimumab
To prevent auto-reactvity > can be through B-cell library: make V gene fragments with high diversity > coupling genotype with phenotype via surface proteins (like antibody) > in phages > screening with immobilized antigen > amplification and analysis > iteration with higher selection procedure: Affinity Maturation > gene isolated eventually for rmAb with high affinity and desired specificity
82
Human antibodies: transgenic mice such as in panitumumab
Mouse embryonic stem cells > make mouse that makes human an d mouse antibodies and mouse with inactivated mouse Ig kappa chain and Ig H chain >> xenomouse made which only makes human IgG kappa/lambda antibodies
83
Types of anti-antibodies
- Anti idiotype: CDR, possible in humanized mAbs - Anti-VH/VL - Anti-glycan - Anti-allotype: specific modifications / polymorphisms in constant Igs - Anti-isotype: in constant parts Ab
84
Anti-isotype anti-antibodies
- Rheumatoid factors (RFs), in RHeumatoid arthritis patients > low affinity > usually IgM > against Fc > no indications that they are induced by the treatment - Anti-OKT3 (mouse mAb) > anti-mouse-Fc made
85
Anti-idiotype anti-antibodies and anti-VH/VL
For chimeric antibodies: determinants outside of the CDRs can be recognized > Idiotype also contains T-cell epitopes
86
Idiotope
Unique determinants of antibody
87
Idiotype
Collection of idiotopes > specific for single clone, includes CDRs
88
Jerne's Network Theory
Unique antibodies made in immune response > recognized by the immune system > little solid evidence for polyclonal antibody responses > regulation of antibody response via anti-idiotype antibodies: to regulate the immune response over time > theory is not true: but treatment with therapeutic mAbs can result in neutralizing anti-idiotype antibodies that may counteract therapy
89
Anti-adalimumab antibodies
> 98% anti-idiotype Test for IL-8 production, induced by TNF > TNF > IL-8 > TNF + ADL > no IL-8 > TNF + ADL + ADA (anti-ADL) > IL-8 >> enriched mutations for CDRs >> standard T-cell mediated response against mAbs >> not a special type of response > Competition for T-cells in GC: all clones bind a but that is unique for adalimumab in SHM during the auto-B-cell maturation > can bind same region to present to T-cells and take up antigen
90
Anti-drug antibodies are always ...
Neutralizing
91
Immune complexes larger than ... may activate the complement
Hexamer > anti-idiotype hexamer against mAb > does not happen practically, antibody can bind one Fab of another one > another one cannot bind to mAb > for complement activation, hexamer bound with Fc tails is required, is not made
92
Drug-induced antibodies are mainly directed to:
Idiotype
93
Antibody engineering: optimalization effector functions > blocking
Minimize effector functions > IgG2/4 > eculizumab (anti-C5) > combi IgG2/3: IgG2 CH1s + IgG4 Fc
94
Antibody engineering: optimalization effector functions > opsonizing
Maximize effector functions > IgG1, IgG3 > glycosylation
95
Antibody engineering: optimalization effector functions > Bispecific antibodies
Duobody for example based on IgG4

Immunology: Research + Clinic (7 decks)

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