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Protein Science > Antibodies > Flashcards

Antibodies Flashcards

1
Q

What are antibodies

A
  • immunoglobulin protein produced as immune defense against foreign agents (antigens)
  • each antibody has a region binding specifically to a particular antigen
  • made of light and heavy chains
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2
Q

Humoral Immune System

A

control of circulating pathogens

  1. B cell binds to antigen. T dependent B cell requires T helper cell.
  2. B cell differentiation + proliferation
  3. plasma cells proliferate and produce antibodies against antibody
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3
Q

Cellular Immune System

A

control of intracellular pathogens

  1. T cell binds MHC-antigen complex activating the T cell
  2. activation of macrophage
  3. CD8 T cell becomes cytotoxic T lymphocyte able to induce apoptosis of target cell
  4. cytokines transform B cells into antibody providing plasma cells
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4
Q

IgG structure

A
  • S-S disulfide bonds connect hinge region and the light/heavy chains
  • constant and variable sequences
  • light chain: one variable and one constant
  • heavy chain: one variable and three constant
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5
Q

Light Chain seuqneces

A
  • 2 domains
  • 214 amino acids in two domains
  • variable sequences have differences in the N terminal domain
  • constant regions are identical in C terminal domain
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6
Q

Heavy chain sequences

A
  • heavy chain is 445 amino acids in 4 domains
  • hinge gives flexibility to bind to antigen
  • 1 variable and 3 constant domains
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7
Q

Ig Fold

A
  • B strand arrangement of B sandwich motif of sheets linked by loops
  • loops are the CDRs with variation
  • gives binding specificity
  • L and H chain fold to yield 3 CDR in each chain to form walls of Ab binding groove
  • therefore there are 3 CDR per subunit and 6 total forming the antigen binding site
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8
Q

Benefits of catalytic antibodies

A
  • high affinity and specific binding of an antibody
  • non-toxic
  • stabilise the TS
  • but enzymes alone cannot be used in treatment : immune response from body
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9
Q

Abzymes

A
  • catalytic Abs having structural complementarity for the TS of an enzyme catalysed reaction
  • strong binding of the TS with high association constant enhancing reaction rate
  • reduce rotational entropy to reduce the free Gibbs energy of the reaction
  • idea is antibody with high substrate affinity yet are able to have catalytic activity-
    Abs bind TS to lower activation energy and enhance reaction rate
  • you need it to target TS and NOT the substrate
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10
Q

Hapten

A
  • small molecule reacting specifically with an antibody but is incapable of stimulating antibody production except in combination with an associated protein molecule
  • hapten in combination with a larger protein becomes antigenic and can elicit and immune response
  • will bind to antibody but if inject won’t provoke them
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11
Q

Cocaine

A
  • alkaloid from coca plant
  • serotonin-norepinephrine-dopamine reuptake inhibitor
  • nervous system stimulator increasing concentrations of the neurotransmitters (not reuptaken)
  • TS hapten approach used to treat overdoses
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12
Q

Cocaine Degradation

A
  • degraded into inactive molecules prior to crossing blood brain barrier
  • hydrolysis at benzoyl ester or the methyl ester
  • non toxic secreted product
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13
Q

Butyrylcholinesterase

A
  • hydrolyses butyrylcholine
  • hydrolyzes cocaine into methyl ester and benzoic acid
  • can hydrolyze toxic compounds containing an ester
  • human esterases are slow to degrade cocaine
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14
Q

BChE Mechanism

A
  1. glutamate H bonds His to form base
  2. His deprotonates Ser-OH group
  3. Ser nucleophilically attacks the ester bond
  4. oxyanion hole stabilised by enzyme backbone
  5. His acts as conjugate acid to donate a proton
  6. release of choline
  7. His deprotonates water
  8. water attacks acetylated Ser
  9. charge delocalisation and oxyanion hole
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15
Q

Cocaine Degradation by Cholinesterase

A
  1. Ser deprotonated by His to make acetylated intermediate
  2. oxyanion hole
  3. negative charge collapse
  4. pick up His proton
  5. release of product intermediate
  6. water deprotonated by His base
  7. nucleophilic attack on Ser
  8. 2nd intermediate and collapse
  9. His proton regenerates Ser active site
    - similar to how the native substrate (butyrylcholine) operates
    - promiscuity in esterase cleavage of ester bond in cocaine
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16
Q

Anzyme Design

A
  • Ab recognises, binds, and catalyses cocaine
  • stablises the TS for better catalysis
  • products released so the Ab is recycled and free : therefore cannot bind tightly
  • to generate Abs with catalytic activity we generate an immune response against molecule mimicking the TS of cocaine degradation
  • hapten must be stable and mimic the TS tetrahedral intermediate structural/electronic properties
  • Hapten elicits Abs stabilising the TS of the reaction
17
Q

TS in Cocaine Hydrolysis

A
  • 1st step of hapten is the generate a cocaine like molecule with TS behaviour
  • intermediate between cocaine and ecogonine methyl ester product
  • can make TS analogs (tetrahedral) that are modified to cause no further catalysis
  • these tetrahedral hapten types elicit hydrolytic antibodies
18
Q

Linker

A
  • 2nd step is after making the TS analogue to conjugate to a larger protein
  • allows exposure of epitope to the Ab
  • allow to induce immune response
  • must determine the linker type, tether site, length
  • linked to a carrier protein : BSA or ovalbumin
19
Q

Link Length

A
  • too short of a linker makes the carrier + phosphonate mono-ester TS analog seen as one identity and the Abs made against BSA not the analog
  • too long of a link means blood proteases cleave the linker before the immune response
  • cleaves the TS/hapten so the Abs are also against the carrier
20
Q

Linker Site

A
  • site is important as you don’t want the linker to interfere with molecular catalysis
  • 3 sites for linker attachment on the phosphonate mono-ester TS analog
21
Q

Linker Attachment

A
  • modifying the lysine amine group to get many hapten molecule attached to increase chances of Ab recognition
  • want more Ab binding a large protein carrier relative to analog
  • this increases hapten antibodies by having a large ratio
  • more accessible sites on the hapten and less on the carrier
22
Q

Catalytic Ab production

A

Conventional:

  • antigen inoculation of mouse
  • remove spleen cells
  • obtain antiserum: mixture of Abs specific for each antigenic determinant

Monoclonal:

  • fuse spleen cells with myeloma cells into hybrid myeloma cells
  • grown on medium selecting the nonfused cells (myeloma cells lack HGPRT and fusion allows alternative nucleotide pathway)
  • obtain individual populations of cloned antibodies
  • screen for catalytic activity
23
Q

Degradation Kinetics

A
  • want a lower Km so you need less substrate to achieve 1/2 Vmax
  • kcat/k0 higher means higher efficiency at low substrate concentrations
  • review*
24
Q

Cocaine Toxicity

A
  • rat survival after infusion of lethal dose of cocaine significantly improved after selected Abzyme administered
  • can it work in humans in vivo?
25
Q

Antibody Catalytic Mechanism

A
  • mechanistically different to esterase (doesn’t bind substrate tightly like normal enzymes)
  • Tyr aligns the binding site for cocaine coordination
  • water molecule activated to attack the ester
  • active site of antibody stabilised tetrahedral intermediate of the oxyanion hole (Tyr-OH)
  • collapse of negative charge
  • product release and Abzyme regeneration
  • products are benzoate and methyl ester
26
Q

Prodrug Activation

A
  • prodrug has no activity and is activated inside the body by the metabolism
  • Abzyme delivering prodrug to a site and activating it or having catalytic antibodies in a specific location activating drugs on arrival
27
Q

Chloramphenicol Abzyme

A
  • raise Abs against hapten

- Ab can catalyse prodrug ester hydrolysis to release chloramphenicol

28
Q

Abzyme Limitations

A
  • haptens fail to generate antibodies catalysing the desired reaction
  • if haptens too closely resemble the final product there is slow product release or inhibition
  • hapten may be difficult to synthesize
  • catalytic efficiency depends on solvent exposed binding site : if too hydrophobic it gets stuck in membrane
  • Hammond Postulate : TS position relative to product/substrate affects the affinity
29
Q

Therapeutic Abs

A
  • murine, chimeric, humanized, human
  • humanised are best as only the CDR loops are grafted onto a human Ab
  • chimeric consists of variable regions derived from a mouse and constant regions derived from human

Protein Science (11 decks)

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