Week 8: Modern Antibody Therapy Flashcards
1
Q
What is polyclonal antibodies?
A
- Bind to various epitopes on a specific antigen
- From many B cells
- Obtained from donation of blood products
2
Q
What is monoclonal antibodies?
A
- Produced by one B cell clone
2.
3
Q
What is monoclonal antibodies?
A
- Produced by one B cell clone
- Bind to a specific epitope on a given antigen
- Same antigen binding site
- More expensive
- No batch to batch variability
4
Q
What secret polyclonal antibodies?
A
Multiple clones of antigen specific B cells
5
Q
How is polyclonal antibodies derived?
A
- An experimental animal or human subject is immunized with Ag one or more times
- Subject is bled and Ab is purified from serum
6
Q
What are the pros of polyclonal?
A
A mixture of Ab directed toward a variety of epitopes are formed, good for agglutination and immunoprecipitation
7
Q
What are the cons of polyclonal?
A
- May produce cross-reactivities against other Ag
- Different bleed points may yield different degrees of affinity
8
Q
What produces monoclonal antibodies?
A
Product of a single stimulated B cell
9
Q
What produces monoclonal antibodies?
A
Product of a single stimulated B cell
10
Q
How is monoclonal antibodies derived?
A
- Fusion of B cells and immortalized myeloma cell
- Technique includes HAT selection medium for fused cells
- Produces mass quantities of single specificity Ab
11
Q
What is passable polyclonal antibodies?
A
Antibodies being passed from one host to another
12
Q
What is convalescent plasma CCP?
A
- Plasma is obtained from recovered individual by plasmapheresis
- Plasma is collected at 14 days past infection who meet strict criteria for donating blood
- Polyclonal antibodies neutralize pathogen
13
Q
What is plasmapheresis?
A
The process of separating plasma from whole blood components
14
Q
What does convalescent plasma CCP treat?
A
- H1N1 influenza
- 2003 SARS CoV 1
- 2012 MERS CoV
- COVID 19
15
Q
What is intravenous immunoglobin (IVIG) used for?
A
- Mostly IgG and varying IgA
- Provides passive immunity through the transfer of antibodies from one host to another
- Cna neutralize a wide range of antigens and superantigens
- Can provide immunoregulatory effects by suppressing functions of effector cells
16
Q
What are therapeutic proteins?
A
- Monoclonal antibodies
- ANtibodies
- Hormones
- Cytokines
- Enzymes
17
Q
How are the characteristics monoclonal antibody production?
A
- mAb’s are large complex proteins
- Contain multiple disulfide bonds
- Glycosylated (post-translational modification)
- Require eukaryotic machinery to produce active form of protein
- Hybridoma technology was developed in 1975
18
Q
How are monoclonal antibodies produced?
A
- Immunize animal with an antigen we want to target.
- Isolate B cells from the spleen of the immunized animal.
- Fuse immunized B cells with myeloma cells (tumor plasma cells) to form a hybridoma which will produce antibodies forever.
- Grow up the cells in a special media that will kill myeloma cells and B cells but will allow hybridoma cells to grow.
- A hybridoma will produce identical antibodies of a single specificity-monoclonal antibodies.
19
Q
What are the approaches of therapeutic antibody development?
A
- Mouse hydridoma
- Phage display
- Transgenic mouse
- Single B cell
20
Q
What are the monoclonal antibody structures?
A
- Murine
- Chimeric
- Humanized
- Fully
21
Q
What is murine mABs?
A
Fully derived from mice
22
Q
What is chimeric mABs?
A
Murine variable region is spiced into the human constant region (70% human)
23
Q
What is humanized mAbs?
A
Murine hypervariable regions (CDRs) spliced into a human antibody (80-95% human)
24
Q
What is fully human mABs?
A
Completely derived from human sequence
25
What is the suffix -cept?
Fab portion has be replaced with a receptor protein sequence
26
What is guidance for industry?
FDS's nonproprietary naming of biological products
27
How are biologics named?
1. Monoclonal antibody core name
2. Distinguishing, nonproprietary suffix
3. Devoid of meaning
4. 4 lowercase letters
28
What are the limiting factors of therapeutic antibodies?
1. Immunogenicity
2. Ability to reach the target tissue and with sufficient concentration
3. Ability to trigger a particular biological effect that would modify a disease process
29
What is immunogenicity?
Ability of a substance to induce an immune response
30
What is immunogenicity good for?
1. An infection but bad for therapeutic antibodies
2. Leads to the production of anti drug antibodies (ADAs) that bind to and neutralize the therapeutic mAB thereby increasing clearance and decreasing efficacy
31
What are immunogenicity?
Contain epitopes that induce an immune response and are also targets of this immune resposne
32
What is reaching target tissue with sufficient concentration?
1. Specificity for the target
2. Antibody affintiy
33
What is triggers biological effects that modify disease process?
1. Blockade of factors that contribute to biological process
2. Antibody mediated cell killing
34
What is the structure of chimeric mAb?
4/12 domains are murine (2Vl and 2Vh)
35
What is the structure of humanized mAb?
murine CDRs grafted into human mAb (IgG)
36
What is the structure of fully humanized mAb?
the protein sequence is fully human
37
What is the structure of pegylated Fab fragments?
polyethylene glycol is attached to the Fab portion of an IgG antibody
38
What is the structure of receptor Fc fusion?
Functional receptor protein joined to the Fc portion of IgG
39
What happens is the mAB is more humanized?
The less immunogenic
40
What occurs when HAMA antibodies?
Increase clearance on mAb and cause hypersensitivity reactions
41
What is pegylation?
Increases circulation retention, protect against enzymatic digestion, slows filtration by kidney, decreases generation of neutralizing antibodies
42
What antibody isotope would make the best therapeutic?
IgG1
43
What are the characterictics of IgG1?
1. Structurally stable
2. Long in vivo half-life
3. Penetrate tissues
4. Induce Fc mediated effector functions
44
What sub isotopes are not desired by ADCC and CDC?
IgG2 and 4, Fab fragments
45
What are the TNF-a inhibiors?
1. Infliximab (Remicade)
2. Certolizumab pegol (Cimzia)
3. Etanercept (Enbrel
4. Adalimumab (Humira)
46
What is Adalimumab (Humira)?
Fully human (IgG1)
47
What is Etanercept (Enbrel)?
(2) TNFR linked to IgG1 Fc
48
What is Certolizumab pegol (Cimzia)?
Pegylated Fab’ fragment
49
What is Infliximab (Remicade)?
Chimeric IgG1 mAb
50
What are the functions of Fc independent ?
Neutralization/atagonism
51
What are functions of Fc dependent?
1. CDC
2. ADCC
3. Opsonization/Antibody Dependent Cellular Phagocytosis (ADCP)
52
What are the targets of neutralization of mAbs?
1. Receptors on the surface of cells
2. Cytokines, growth factors secreted by cells
53
What are the functions of anti-cytokine mAb?
1. Bind to the cytokine preventing the cytokine from binding to its cytokine receptor and sending a signal
2. Bind to cytokine receptor preventing cytokine from binding to the active site and sending a signal
54
Why is mAb location important?
Determine how effective the antibody will be at preventing biological signaling
55
What are the drugs that block HER2 through the ErbB receptor?
Trastuzumab (Herceptin) and Pertuzumab (Perjeta)
56
What is Bamlanivimab (IgG1k) used for?
Against the spike protein of SARS-CoV-2 and blocks attachment to the human ACE2 receptor
57
What is Etesevimab (IgG1k)
Against the spike protein of SARS-CoV-2 with amino acid substitutions in the Fc region to reduce effector function.
58
What is the function of both Bamlanivimab and etesevimab?
1. Bind to different but overlapping epitopes in the receptor binding domain (RBD) of the Spike protein.
2. co-administered in approved individuals with mild to moderate COVID-19
3. IV infusiotn
4. Emergency use
59
What is the use of mAb for COVID?
Neutralization by binding to SARS-CoV-2 preventing viral attachment and entry into cells
60
How do we control T cell activation?
1.When targeting tumor cells, we want to increase T cell activation to cause cell death. Negative costimulatory proteins are blocked by mAbs to allow T cell activation.
2. In autoimmune conditions, we want to decrease T cell activation and this prevent inflammatory cytokine production.
61
What is the overview of antibody mediated effector mechanisms?
1. All effectors of the mechanisms require the presence of the Fc portion of the antibody
2. Therapeutics contains only the Fab portion may block signaling without inducing these mechanism
62
What are the mechanisms for cell death?
1. CDC
2. ADCC
3. ADCP
63
What is CDC?
1. Fc region of mAb can recruit effector cells such as NK cells, macrophages or neutrophils to activate complement mediated cell death by cell lysis
2. Activation of complement causes release of C3a, C4a, and C5a and cause inflammation
64
What is ADCC?
1. Fc region of mAb can recruit effector cells such as NK cells.
2. NK cells form an immunological synapse with antibody labeled target cell.
3. NK cells release granular contents to induce apoptosis.
4. Apoptosis is a less inflammatory form of cell death.
65
What is ADCP?
1. Fc receptor dependent form of cell death.
2. Macrophages and neutrophils have FcRs that recognize the Fc portion of the IgG mAb.
3. This triggers the phagocytosis of the antibody coated target cell.
4. Referred to as ADCP when it is a tumor cell or altered host cell instead of opsonization.
66
What are biosimilars?
Same aa sequence as original mAb bu produced from a different clone and can have different glycosylations
67
What are the modifications of biosimilars?
1. Improve binding affinity (CDR regions)
2. Reduce immunogenicity
3. Influence the Fc mediated effector functions
68
What are the side effects of monoclonal Ab?
1. Risk of immune reactions
2. Acute anaphylaxis (infusion reaction, IgE mediated)
3. Serum sickness
4. Generation of anti drug antibodies
5. Acute infusion reactions
6. Risk of infection and malignancy
69
How can mAb thrapy cause immune reactions?
1. Due to immunogenicity of mAB
2. More prevalent with murine mAb
70
How can mAb therapy cause serum sickness?
More common in chimeric mAb
71
How can mAb therapy cause generation of anti drug antibodies?
1. All mAbs will cause the generation of anti-drug antibodies with repeated administration.
2. Anti-drug antibodies are more common with more immunogenic mAbs
72
How can mAb therapy cause risk of infection and malignancy?
1. mAbs against TNFα have been associated with reactivation of latent tuberculosis, as well as with other serious infections and malignancies.
2. Development of an acquired immunodeficiency
73
What is Enzyme-linked immunosorbent assay (ELISA)?
Assays that use antibodies or antigens covalently bound to enzymes
74
What is indirect ELISA?
1. Detects presence and concentration of Ab in a sample
2. Method of choice to detect presence of serum Ab against HIV, HBV, HCV
75
What is sandwich ELISA?
1. Measures Ag presence and levels
2. Useful for measurement of soluble cytokine concentrations
76
What is competitive ELISA?
1. Measures amount of Ag in a sample
2. The more Ag in original sample, the lower the final signal
77
What is Lateral Flow Immunoassay used for?
Used to detect presence of antigen or antibody in clinical sample
78
What are analyses?
urine, saliva, sweat, serum, plasma, blood, mucous
79
How is lateral flow immunoassay
1. Liquid sample is placed on absorbent pad and carried by flow of buffer added to pad.
2. Sample interacts with antibodies that are specific for the target analyte and are conjugated to colored particles–most commonly colloidal gold
3. Test line contains mAbs to analyte
4. Control line contains mAb to species of mAb-colloidal gold or to colloidal gold
80
What is hemagglutination?
Used to detect any Ag conjugated to the surface of RBCs
81
What occurs during an agglutination reaction?
1. Antibodies that can bind a surface Ag will cross-link the RBCs, forming a large clump that doesn’t settle in the plate or test tube
2. Routinely used in blood typing- Coombs Test
3. Can be adapted to measure levels of Ab in a sample directed against any Ag that can be attached to the surface of an RBC
