Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Immunology > Immunotherapy > Flashcards

Immunotherapy Flashcards

1
Q

how do antibodies protect against infection?

A

they act as specific labels for infectious material, and the material can then be eliminated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

how are antibodies used in research and medicine?

A
  • Antibodies are widely used in research, as diagnostics and increasingly as a new class of therapeutic drugs
  • Bind to anything that’s foreign to be used as labels
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what are the useful properties of antibodies for research and medicine?

A
  • Diverse >10^8 specificities
  • Specific, high affinity KD 10^-8 – 10^-9 M
  • Domain structure is stable which facilitates protein engineering
  • Multivalent improved binding, cross-linking can be useful
  • Effector properties useful in some techniques, therapeutics
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

how is antisera produced in research?

A
  • Make antibodies by immunising an experimental animal to induce secondary/memory responses and IgG production
  • Producing antisera (serum containing antibodies to target antigen
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

how are antibodies used as tags in research?

A
  • Antibodies can be purified and labelled with a detectable tag e.g. a fluorescent probe, an enzyme or even gold particles
  • Antibodies are generally used to identify and label molecules in complex mixtures
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what kinds of research techniques can antibodies be used in?

A
  1. Immunofluorescence, FACS - fluorescent tag
  2. ELISA, immunoblotting, immunohistology
    - enzyme–>coloured product
  3. radioimmunoassay imaging - radioisotope
  4. immuno-electronmicroscopy - gold particles
  5. affinity purification, immunoprecipitation - sepharose
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

what part of the antigen does antibody recognise?

A

the epitope

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what are the two types of epitope?

A
  1. Linear – adjacent in sequence (non-conformational) linked to primary structure of the antigen
  2. Discontinuous – non-adjacent (conformational) distant to primary structure of the antigen but when folded they come in close proximity
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

how many epitopes does an antigen have?

A

large antigens can have several epitopes, which can be different or repeated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

can antibodies bind to multiple epitopes on an antigen?

A

yes, antibodies can bind monovalently to single epitopes on an antigen or multivalently to repeated epitopes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

how can antigens have different or repeated epitopes?

A

different:
- an antigen may have 4 different epitopes where each can be recognised by 4 different antibodies

repeated:
- an antigen may have repeating epitopes where an antibody can bind multivalently to them

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what is immunogenicity?

A

Immunogenicity: ability of an antigen to induce an immune response

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what affects the immunogenicity of an antigen?

A
  • its foreigness
  • its molecular size
  • its chemical composition
  • its ability to provoke T cell responses
  • its role as an adjuvant
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

how does an antigen’s foreigness affect its immunogenicity?

A

sequence homology between antigen and equivalent protein in recipient
- Rabbit, sheep and mice are used to make antibody for human, so their antigen must be different enough from human
- prevents the antibody from recognising body tissues

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

how does an antigen’s molecular size affect its immunogenicity?

A

Molecular size - <1000 daltons so can be lost from the body easily
- carrier proteins can help increase the molecular weight so the antigen can stay in the body for longer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

how does an antigen’s chemical composition affect its immunogenicity?

A

Chemical composition – aromatic groups, charged residues
- Some non-covalent interactions are stronger than others

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

how does an antigen’s ability to provoke T cell responses affect its immunogenicity?

A

T cells aren’t good at responding to small molecules, so can link the antigen to carrier protein for an enhanced T cell recognition

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

how does an antigen’s role as an adjuvant affect its immunogenicity?

A

if it has adjuvant activity, it can induce inflammation and danger signals, increasing its activation of the immune response

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what is conventional antisera?

A

the generation of polyclonal antibodies
- antisera is the product of several B cell clones - mixture of antibodies specific to different epitopes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

what is a polyclonal antibody?

A

antibody that is specific to different antigen epitopes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

how are polyclonal antibodies generated?

A

Rabbits, sheep, horses are used
- Antigen has different epitopes on its surface which can induce formation of antibodies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

what are the advantages of polyclonal antibodies?

A

relatively cheap, robust (may recognise partially denatured/unfolded antigen)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

what are the disadvantages of polyclonal antibodies?

A
  • specific for multiple epitopes (may not be specific for antigen of interest if it shares epitopes with other proteins),
  • need pure antigen to immunise,
  • can be difficult to standardise (different animals will respond differently to the same protein)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

what is cross-reactivity of polyclonal antibodies?

A

A, B, C – different epitopes
- Antiserum specific for Antigen 1 CROSS-REACTS with Antigen 2
- Problem when we want an antibody specific for a single epitope

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

what are monoclonal antibodies?

A

Antibody specific for a single antigen epitope, derived from single B lymphocytes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

how are monoclonal antibodies produced?

A
  1. Took B cells from mouse and immortalised it by fusing it to immortal cancer cell line – used myeloma cells which cannot produce antibodies
  2. Lymphocytes taken from immunised animal and fused with myeloma cell line with polyethylene glycol (PEG), induces cell:cell fusion
    - Forms fused B cells and myeloma cells, but some myeloma and B cells are unfused
    - Unfused B cells die anyway in culture
    - Select against unfused myeloma cells
    -unfused mutant myeloma cells lacking enzyme HGPRT (hypoxanthine-guanine phosphoribosyl transferase) needed for purine biosynthesis, so die in selective HAT (hypoxanthine-aminopterin-thymidine) medium
    - any fused cells have HGPRT enzyme from B cell parent, so these survive
  3. fused cells are grown up – hybridomas
  4. Cells are cloned = grow up from single cells
  5. Check that they make antibody against the antigen, can isolate and purify the monoclonal antibody
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

what are the advantages of monoclonal antibodies?

A
  • highly specific,
  • can be standardised (can be grown indefinitely),
  • pure antigen not needed for immunisation
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

what are the disadvantages of monoclonal antibodies?

A
  • conformation sensitive
  • expensive
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

what are the uses of monoclonal antibodies?

A
  • used in therapy
  • used to identify cell surface molecules
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

how can monoclonal antibodies be used to identify cell surface molecules?

A

human leukocytes -> CD (cluster of differentiation) is a classification system to help identify which antibodies recognise which molecule. (>300)
*- can use monoclonal antibodies to identify cell types e.g. all T cells express CD3, or T cell subpopulations e.g. CD4 and CD8
- can be used to see what stage of differentiation a cell is at

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

what is the issue with using monoclonal antibodies from animals in therapy? how can this be overcome?

A

Rodent antibodies induce immune responses in human patients e.g. HAMA (Human Anti Mouse Antibody)
- Mouse antibodies are recognises by our body as foreign via HAMA
- Can result in serum sickness

generation of human antibodies for therapy

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

how can human antibodies be generated?

A

Antibody engineering:
1. Antibody chimeras:
2. Humanised antibodies:

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

how are antibody chimeras made antibody engineering? are these useful for therapy?

A

Antibody chimeras: Splice mouse V region genes and fuse to human C region genes.
- Problem: mouse V regions still immunogenic so recognised as foreign by body

34
Q

how are humanised antibodies generated in antibody engineering? are these effective?

A

Humanised antibodies: Splice human framework region genes and keep mouse CDR region genes
- a.k.a CDR grafting
- facilitated by stable immunoglobulin domain structure
- disadvantages: may lose affinity/specificity, time-consuming.

35
Q

what strategies can be used to generate fully human antibodies?

A
  1. transgenic mice
  2. antibody gene libraries
  3. single B cell antibodies
36
Q

what are antibody gene libraries?

A

-Antibody V genes are cloned from naïve/immune B cells using a suitable vector and used to make a large “library”.
-The antibodies in the library can be expressed in bacteria or on the surface of bacteriophage. Antibodies against the desired antigen are selected from the library, usually using phage display techniques.

37
Q

how can transgenic mice be used to generate human antibodies?

A

TRANSGENIC mice expressing human immunoglobulin genes.
- Mouse antibody genes replaced by human antibody genes (“Xenomouse”).
- Mice can be immunized to generate human antibodies by conventional monoclonal techniques.

38
Q

how are antibodies derived from gene libraries?

A
  1. Isolate mRNA from antibody producing cells.
    - Blood, lymphoid tissue, bone marrow
  2. Reverse transcribe mRNA. Amplify Fab or V region cDNA by PCR
  3. Clone and express Fab or V region cDNA in bacteria/phage (phage display) to produce an antibody library
    - Phagemid vectors – express soluble protein in bacteria or on surface of filamentous phage particles (M13)
    - V genes are fused to bacteriophage coat protein gene using phagemid vector
    - gene library expressed as V genes on surface of phage
  4. Screen antibody phage display library vs solid phase antigen “panning” to select desired antibody
    - Coat antigen onto surface, put phage on, and the ones that bind and aren’t washed away can be selected
    - Can harvest the specific phage and regrow in bacteria to enrich for antigen binding regions
39
Q

how are antibodies selected for by phage display?

A

Following selection by binding to antigen, phage are used to re-infect bacteria and the process repeated to enrich for antigen binding V regions.
- V region genes can be spliced to C region genes and expressed as whole antibodies in mammalian cells (to maintain correct glycosylation etc.)

40
Q

how can synthetic human antibody gene libraries be made without immunisation?

A
  1. Clone human V region genes + “randomised” CDR regions → formed libraries with > 10^9 members
  2. Randomise parts of the molecule to interact with CDR = enhance variability
  3. Use antigen to select phage that bound
  4. Phage CDRs can be further mutated to improve specificity/affinity and repeat selection process – mimics AID enzyme which does somatic hypermutation

NO NEED TO IMMUNISE

41
Q

how can single B cell antibodies be used to generate human antibodies?

A
  • Single antigen-specific B cells from patient blood or lymphoid tissues are isolated using e.g. fluorescent antigen and fluorescence-activated cell sorting (FACS).
  • B cells that bind the fluorescent antigen are selected
  • Expressed antibody V genes are amplified and cloned into phagemid vector which can be cloned and screened
  • Useful for generating antibodies against emerging pathogens e.g. covid
42
Q

what are nanobodies?

A

single domain antibodies:
- Just composed of heavy chains
- Nanobody has single variable immunoglobulin domain which binds antigen tightly
- Chemically very stable
- Easily expressed in bacteria, yeast, as they have just one domain
- Humanisation feasible as it has one domain

43
Q

what formats of monoclonal antibodies are used in therapy?

A
  • fully mouse
  • chimeric
  • humanised
  • fully human
44
Q

how were antibodies used in therapy as passive immunisation? what was the drawback?

A
  • Emil Adolf von Behring (1880s) showed serum from immunised animals was an effective treatment against diphtheria and tetanus.
  • Decrease in popularity due to side-effects (serum sickness) and discovery of antibiotics
45
Q

how are monoclonal antibodies used in therapy?

A

Slow uptake but some human monoclonal antibodies are now being used to prevent/treat infection e.g. respiratory syncytial virus (RSV), Ebola, COVID

46
Q

is conventional antisera still used in some therapies?

A

yes, conventional antisera or human IgG preparations (IVIg) still used in some therapies:
- Neutralise toxins e.g. anti-snake venom (antisera prepared in animals), anti-tetanus (pooled human IgG)
- Treat infections caused by emerging pathogens e.g. convalescent sera to treat Ebola and COVID

47
Q

what are examples of the use of monoclonal antibodies in cancer?

A

Anti-CD52 antibodies (CAMPATH)

Anti-CD20 antibodies (Rituximab)

Anti-Her2 antibodies (Herceptin)

antibodies can act as magic bullets to target cancer

48
Q

how is anti-CD52 (CAMPATH) used to treat cancer?

A

Anti-CD52 antibodies (CAMPATH) – CD52 found on all leukocytes
- Recognises leukocytes, good activator of complement and ADCC
- Use in leukaemias, lymphomas – shrinks tumours
- First humanised (IgG1) antibody used in clinical trials

49
Q

how is anti-CD20 (Rituximab) used to treat cancer?

A
  • Recognises B cells, good activator of complement and ADCC
  • Used in leukaemias, lymphomas
  • Chimeric antibody
50
Q

how is anti-Her2 (Herceptin) used to treat cancer?

A

Recognise Her2 (receptor tyrosine kinase) expressed at high levels in 25% breast tumours, invokes ADCC

51
Q

how can antibodies be used therapeutically?

A
  1. Depletion of leukocytes
    - E.g. antibodies of CD52 (CAMPATH), CD3, CD4
    - Organ transplantation, treat graft versus host disease, treat autoimmune disease
    - GVHD – antibodies from kidney graft may recognise patient as foreign and attack
  2. Blocking of cytokines, cytokine receptors, soluble mediators
    -E.g. antibodies to dampen down cytokines: TNF-alpha, IL-1, IL-6, complement protein C5 (or their receptors)
    - Treat inflammatory/autoimmune disease
    - Treat allergy e.g. antibodies to IgE, cytokines
    - Treat over-reactive response to some infections e.g. COVID
  3. Immune checkpoint inhibitors
    - E.g. antibodies to CTLA-4, PD-
    - Cancer immunotherapy
52
Q

how do cancers induce immunesuppression?

A
  1. they produce cytokines such as IL-10 which activate Tregs
  2. cancer induces expression of CTLA-4
  3. cancer expresses a PD-ligand
53
Q

what is CTLA-4?

A

CTLA-4 induced on activated T cells;
- CTLA-4 has higher avidity for B7 than CD28, but induces inhibition of T cells
- expressed by cancers

54
Q

what is PD-ligand?

A

PD-1 (transiently expressed on activated T cells) interacts with ligand PD-L and induces inhibition of T cells

55
Q

how does cancer exploit CTLA-4 and PD-1?

A

CTLA-4 and PD-1 act as immune checkpoints to dampen down immune response
- These are exploited by cancer cells to evade the immune system
- Antibodies that block inhibitory immune checkpoints can reverse immunosuppression

56
Q

give examples of antibodies which reverse the immunesuppression of cancer:

A

Nivolumab binds PD-1, blocking its interaction with PD-L = T cell is reactivated

Ipilimumab binds CTLA-4, blocking its interaction with B7 = CD28 can now interact with B7, so T cell is reactivated

Antibodies that block immune checkpoints have shown beneficial effects in metastatic melanoma and some types of lung cancer – cancer therapy by inhibiting negative immune cell regulation

57
Q

how can antibodies be used in immunotherapy?

A

Used to exploit natural antibody binding/effector functions e.g. ADCC
- Cancer cell has molecules on its surface that are unique, so antibodies that target these can induce NK cells to kill the tumour

58
Q

what must be ensured when using antibodies for immunotherapy?

A

Important to choose subclass of IgG with appropriate effector functions for intended therapy:
- Complement activation: IgG3 > IgG1 > IgG2
- Fc receptors on phagocytes: IgG1=IgG3>IgG4
- Fc receptors on NK cells: IgG1=IgG3
- Promote interaction FcRn to increase antibody half-life): all subclasses

59
Q

how can antibodies be improved for therapy?

A

by mapping effector sites on the antibodies that are responsible for activating innate immune processes and enhancing them through mutation

60
Q

what effector sites were mapped on antibodies?

A

mapping effector sites on antibodies:
1. C1q binding interact with hinge in CH2 domains
- Hinge/C-gamma2 domains

  1. Fc-gammaRI, Fc-gammaRII interact with “Lower hinge” – interact with phagocytes
    - CH2 antibodies are bent
  2. Fc-gammaRIIIA for NK cells
  3. Fc-gammaRn – could mutate these to improve antibody function
    -half-life

these regions could all be mutated to improve antibody function

61
Q

why is precise mapping of effector sites useful in improving antibodies for therapy?

A

Precise mapping of effector sites can be used to generate “designer antibodies” for use in therapy.
- Effector functions in CH2 domain – important for defining role of antibody

62
Q

what methods can be used to improve antibodies?

A
  1. glycosylation
  2. protein/glyo-engineering
  3. label antibody with drug/prodrug/toxin/radionuclide
  4. using antibody fragments
  5. generation of bi-specific antibodies
63
Q

how can glycosylation improve antibodies? are there limitations?

A

IgG has 2 N-linked glycans (Asn 297) which holds CH2 domains apart -carbohydrate is important for effector functions as it maintains the conformation of IgG
Glycoengineering:
- Removal of fucose improves IgG interaction with FcγRIII (the FcR on NK cellsfor ADCC)
- PEGylation especially important for antibody fragments.

However, it may reduce immunogenicity

64
Q

how can protein/glyo-engineering improve antibodies? are there limitations?

A
  • Antibody engineering to improve half-life (enhance FcRn interaction), improve or remove effector functions (e.g. enhance ADCC, complement activation).
  • Alterations to the glycosylation of IgG can improve interaction with FcRn (increases half-life) and interaction with FcR on NK cells (enhances ADCC)
65
Q

how can labelling improve antibodies?

A

Label antibody with drug/prodrug/toxin/radionuclide:
- tumour-specific antibody conjugated to drug
- antibody-drug conjugate binds and is internalised, killing the tumour cell.

66
Q

how can use of antibody fragments improve the use of antibodies in therapy?

A

For some applications, antibody fragments are useful – better tumour penetration
- e.g. Fab fragments, single-chain Fvs (scFv) – paired V regions, linked by peptide
- difficult for whole antibody to get into a large tumour, so using small fragments of the antibody is useful

67
Q

how can bi-specific antibodies be useful in therapy?

A

Bi-specific antibodies with dual specificity can link tumour binding to effector cell proteins e.g. tumour antigen + CD3
- Antibody can bind to CD19 protein on B leukaemia cell and also bind to CD3 on T cells to induce cell-mediated immune response

68
Q

what is convalescent sera? is it effective against disease?

A

Convalescent sera (blood taken from recently infected patient): limited efficacy (polyclonal, time after infection critical), limited supply, stringent safety requirements

69
Q

what monoclonal antibody targets SARS-COV-2 for passive immunisation?

A

Sotrovimab
- engineered to possess an Fc Lysine-Serine mutation (M428L/N434S) that confers enhanced binding to the neonatal Fc receptor resulting in an extended half-life and potentially enhanced drug distribution to the lungs
- However covid mutates quickly so this may have temporary effectiveness

70
Q

what are the advantages of monoclonal antibodies as passive immunisation for covid?

A
  • immediate protection (for up to 4 weeks)
  • suitable for immunocompromised patients
  • may relieve severe symptoms
71
Q

what are the disadvantages of monoclonal antibodies as passive immunisation for covid?

A
  • no long-lasting protection
  • not very effective against latest variants
  • may promote “escape mutants”
  • expensive to produce and administer
72
Q

how may monoclonal antibodies be used to treat covid in the future?

A
  • antibodies to conserved epitopes
  • “cocktails” of antibodies to different targets or bi-specifics
73
Q

what monoclonal antibodies are used to treat excessive inflammation in covid?

A

anti-interleukin 6 receptor (Tocilizumab)

anti-C5a (Vilobelimab) – prevent complement activation and associated inflammation

74
Q

what is CAR-T cell therapy?

A

Chimeric antigen receptor T cell therapy:
- T cells engineered to recognise a tumour antigen
- T cells harvested from blood of patient and engineered to target tumour, and then reinjected into patient to attack that tumour

75
Q

give an example where CAR-T cell therapy has been used to treat disease:

A

Acute Lymphocytic Leukaemia (B cell cancer):
- CD19 – antigen expressed at high levels by leukaemia cells
- T cells are isolated from tumour and engineered in vitro to express chimeric antigen receptor (variable regions of antibody) to CD19
- Chimeric antigen receptor = anti-CD19 (scFv) fused to signalling domains.
- Signalling domains contain ITAM motifs to bind to CD19
- T cells are then reinjected to patient and target the tumour

76
Q

what are the advantages of CAR-T cell therapy?

A
  • Expression of CAR and activation of T cells in vitro overcomes requirement for MHC recognition
  • CAR-T therapy is beneficial in treating some forms of leukaemia.
  • Recently also used to treat systemic lupus erythematosus (SLE).
  • CAR-T cells attack self-reactive B cells
77
Q

how may immunotherapies be used in the future?

A
  • Soluble T cell receptors (e.g. Kimmtrak)
  • Cancer vaccines
  • Tumour infiltrating lymphocyte therapy (TIL)
  • Gene editing of antibody genes in vivo (CRISPR-Cas9)
  • Modulating innate immune cells
  • Use of γδ T cells for cancer
78
Q

how can soluble T cell receptors be used in cancer therapy?

A

“Kimmtrak” a recently approved drug for the treatment of uveal melanoma
- Soluble “affinity matured” TCR that recognises peptide from a tumour antigen (gp100) highly expressed by melanoma cells fused to a scFv anti-CD3 antibody.
- TCR will bind to tumour cell due to anti-CD3 antibody
- Requires MHC as it is a TCR

79
Q

what is the advantage of using TCRs in cancer therapy?

A
  • Does not require engineering of patient’s cells
  • Permits targeting of intracellular antigens
80
Q

give an example of where immunotherapy has went wrong?

A

Clinical trials of drug TGN1412
- Developed by Tegenero for treating autoimmune disease.
- within hours of drug administration, multiple organ failures in all volunteers
- Most immunosuppressive antibodies block activity, whereas TGN1412 binds to CD28 on naïve T cells, inducing stimulation.
- Animal studies indicated selectivity for naïve suppressor T cells BUT in humans memory effector T cells in tissues also express CD28.
- Cytokine storm
- TGN1412 was also an IgG4 antibody, assumed not to interact with FcR

Immunology (8 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions