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Infection and Immunity > Antibody Engineering > Flashcards

Antibody Engineering Flashcards

1
Q

How many specificities does the antibody repertoire have?

A

10^9

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2
Q

What are hypervariable regions?

A

Also called complementary determining regions
Most variable part of the Ab
Form a complementary surface for antigen to allow binding

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3
Q

What is affinity?

A

Binding strength for single paratope-epitope interaction - intermolecular non-covalent interactions

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4
Q

What is avidity?

A

Combined strength of multiple synergistic paratope-epitope interactions
e.g. pentameric IgM with 10 antigen binding sites has high avidity

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5
Q

Describe antibody-dependent cell-mediated phagocytosis

A

Fc receptor on phagocyte binds antibody with antigen bound to it
This induces the phagocytic cell to phagocytose the pathogen

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6
Q

Describe antibody-dependent cell-mediated cytotoxicity

A

Antibody bound to an antigen on a pathogen
This binds to Fc receptor on NK cell
Stimulates NK cell to kill the pathogen

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7
Q

What process is independent of Fc receptors?

A

Complement fixation via classical pathway
Antibody bound to pathogen
First part of complement system binds to the antibody and eventually lead to killing of the pathogen

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8
Q

What are polyclonal antibodies?

A

Antibodies with affinity for the same antigen but different epitopes of that antigen

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9
Q

What is the polyclonal antibody response?

A

Mouse injected with antigen with different epitopes
Mouse contains different B cells each with a BCR that binds a specific antigen
When the B cells are exposed to the epitopes on the Ag, the B cells with specificity for the epitopes present will undergo clonal selection and they will divide and proliferate to form a clone of cells
All these cells will produce the same antibody with the same specificity but for different epitopes of the antigen

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10
Q

How are monoclonal antibodies made?

A

Take the antigen we want the antibody for and inject into a mouse
Allow mouse’s immune system to response
Harvest the B cells from the mouse and in culture using polyethylene glycol fuse the B cells to myeloma cells
Select hybridoma cells (fused cells) using hypoxanthine-aminopterin-thymidine (HAT) selection
Dilute the hybridomas so we have single cells
Culture the single cells - they will then divide and form a clone which will then produce antibodies
These antibodies will all be of one type as they have come from 1 clone of cells so they are mAbs

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11
Q

What are myeloma cells?

A

Immortal cells derived from B cell tumour but do not produce antibodies of their own
Lack hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene

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12
Q

How does HAT selection occur?

A

Aminopterin stopes production of DNA synthesis by normal pathway in cells
Second way of making DNA called salvage pathway - requires the HGPRT gene
This means all the myeloma cells die because they do not have that gene
All the B cells die because they are naturally short lived
This leaves the hybridomas

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13
Q

What are problems with hybridomas?

A

Low yielding - do not produce many Abs
Genetically unstable

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14
Q

How were the limitations of hybridomas overcome?

A

Isolate the genes that make the mAb work (mAb genes)
Clone these genes and put them in other cells - expressed as a recombinant antibody in another expression system

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15
Q

What does cloning of the mAb genes allow?

A

Further genetic manipulation which is the basis of many antibody engineering techniques

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16
Q

What are some problems with using mammalian cell cultures for mAb production?

A

Expensive
Very low scale up capacity - cannot produce in large amounts

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17
Q

What are alternative expression systems for mAb production?

A

E.coli
Yeasts - IgG has been prod in Pichia pastoris
Transgenic plants - IgG, secretory IgA
Transgenic animals

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18
Q

Describe mAb production in E.coli

A

Can produce whole Abs but they are not fully functional as E.coli lacks enzymes for glycosylation
Fc does not interact with complement

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19
Q

Describe mAb production in transgenic animals

A

Expression in mammalian milk gland
Efficiency of transformation is low
Production of functional IgG reported in goats

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20
Q

Give an example of humanisation in mAb engineering

A

First licensed mAb used in human therapy was called Orthocline OKT3 - a mouse mAb IgG2A prod in hybridomas
Was used to prevent kidney transplant rejection
However not used as much now due to side effects being reported - patients often get human-anti-mouse ab response (HAMA)

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21
Q

What are the different types of mAbs?

A

Xenogeneic - 0% human
Chimeric - 66% human
Humanised - 90-95% human, CDR grafted
Fully humanised - 100% human, fully human mAbs can be prod using transgenic mice

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22
Q

How are fully humanised mAbs produced in trasngenic mice?

A

Endogenous mouse IgG genes are deleted and replaced with human IgG genes
After immunisation, mouse B cells make human antibodies
Human mAbs can then be made using the hybridoma method of production

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23
Q

What is trastuzumab (herceptin)?

A

Humanised IgG1 produced in mammalian Chines hamster ovary (CHO) cells

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24
Q

What does herceptin bind to?

A

Binds to receptor ERBB2, a tyrosine kinase

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25
Q

What is herceptin used for?

A

Used to treat metastatic breast cancer that overexpresses ERBB2 receptor
Overexpression occurs in 25-30% of early-stage breast cancers

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26
Q

What does ERBB2 do?

A

Involved in transmitting signals leading to cell proliferation
During cancer it transmits constitutively, leading to uncontrolled survival, proliferation and angiogenesis

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27
Q

What does herceptin do?

A

Downmodulates ERBB2 inhibiting signalling pathways
Inhibits angiogenesis
Induces ADCC
Sensitises cells to chemotherapy

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28
Q

What is eculizumab (soliris)?

A

Humanised mAb engineered to have a hybrid IgG2/IgG4 Fc region
Produced in mammalian cell line NS/0

29
Q

What is eculizumab used for?

A

Used to treat paroxysmal nocturnal globinuria (PNG) - a rare condition in which the RBCs are destroyed by the complement system

30
Q

What does eculizumab do?

A

Binds to complement component 5, inhibiting the cascade
The hybrid Fc region cannot bind to Fc receptors or activate complement

31
Q

How are antibody fragments produced as part of mAb engineering?

A

Using recombinant DNA techniques

32
Q

What are two commonly produced fragments?

A

Fab and single chain Fv (scFv)

33
Q

How is the Fab fragment produced?

A

Vh and Ch1 region along with VL and CL region put into an expression system
The DNA of these is transcribed and translated
Proteins for those regions are made
Inside the cell these proteins will fold to form the Fab region that can then bind to an antigen

34
Q

How is the scFv fragment produced?

A

DNA codes for the Vh and VL regions with a linker sequence, L, between them
When the DNA is transcribed and translated, the protein will fold to form a single chained antibody which will bind the antigen

35
Q

Why is the scFv fragment useful?

A

Because only one chain is involved in producing the fragment, meaning there is no need for 2 different chains to associate within the cell to form the fragment

36
Q

What are some considerations when choosing whether to use mAb or antibody fragment?

A

Method of production - e.g. if using E.coli as an expression system, to bear in mind that this system cannot produce full-length, fully functional antibodies
Avidity - number of binding sites; in a full length antibody there will be 2 binding sites so higher avidity
Fc effector function - is the Fc region required? If it is then full length antibody needed as Fc region involved in ADCC, ADCP, complement
Target tissue - e.g. if the target tissue is a tumour then scFv may be able to penetrate more effectively than a full-length Ab as it is smaller
Clearance - antibody fragments usually cleared from the body faster than full-length Abs; longer interaction with target tissue may be wanted but it can cause a problem in some therapies

37
Q

What is abciximab (ReoPro)?

A

A chimeric Fab fragment (part mouse and part human) that binds to glycoprotein IIb/IIIb receptor on platelets
Produced in mammalian SP2/0 cells

38
Q

What is ReoPro used for?

A

Used in coronary artery operations to prevent platelets sticking together and causing blood clots
Fc region not required hence why Fab fragment is used and not full-length Ab

39
Q

What are conjugated Abs?

A

A mAb coupled with an effector molecule to enhance the function of the mAb
e.g. when targeting a tumour, cytotoxic drug can be attached to the mAb to kill the tumour

40
Q

What are some effector molecules that can be coupled to mAbs?

A

Plant/bacterial toxins
Enzymes
Radionuclides
Cytotoxic drugs

41
Q

What is chemical coupling?

A

Method commonly used to conjugate antibodies with effector molecules
Usually done with the aid of chemical ligands (joining molecules)

42
Q

What are sites for coupling on the mAb?

A

Usually thiol groups (-SH, cysteine), amine (-NH2, lysine) or carbohydrate

43
Q

How can attachment sites be added into a mAb?

A

Using self-directed mutagenesis (although the function of the mAb must not be impaired)

44
Q

What is SMCC attachment?

A

Form of chemical coupling - stands for succinimydl 4-N-maleimidomethylcyclohexane-1-carboxylate (SMCC) attachment

45
Q

What is another method of coupling?

A

Genetic engineering for protein effector molecules

46
Q

How can scFv be coupled to a toxin?

A

Single chain DNA joined to DNA coding for toxin
Whole chain is expressed and protein forms

47
Q

What is ibritumomab tiuxetan (Zevalin)?

A

Mouse IgG1 produced in mammalian CHO cells
Labelled with yttrium-90 (a beta radion emitter)

48
Q

What does zevalin do?

A

Binds to CD20 expressed on surface of B cells, function unclear
Used to treat non-Hodgkins lymphoma
Induces cell death by radiation
May possibly elicit ADCC, CDC, apoptosis

49
Q

What are bispecific antibodies?

A

Antibodies that bind to 2 targets

50
Q

How do bispecific antibodies bind?

A

One arm binds to CD19 on B cell tumour
Other arm binds to CD3 on effector T cell
This brings the effector T cell close to the tumour, enabling killing of the tumour

51
Q

How do bispecific antibodies bind?

A

One arm binds to CD19 on B cell tumour
Other arm binds to CD3 on effector T cell
This brings the effector T cell close to the tumour, enabling killing of the tumour

52
Q

How can bispecific antibodies be produced?

A

Chemical cross-linking of IgG, Fab of scFv fragments
A hybrid hybridoma - 1 hybridoma that produces 1 antibody and another hybridoma that produces another antibody; fuse these together and a proportion of the Abs prod will be hybrid and these can be selected

53
Q

What is catumaxomab (Removab)?

A

Rat IgG2b/mouse IgG2a bispecific antibody produced in a hybrid hybridoma

54
Q

What does Removab do?

A

Binds to epithelial cell adhesion molecule (EpCAM) adn CD3 antigens
Approved for removal of tumour initiating cells in the ascites in patients with EpCAM-positive cancer if standard therapy not available
Recruits cells from adaptive and innate immune system

55
Q

What are ascites?

A

Accumulation of fluid in the abdominal cavity

56
Q

What is glycoengineering in mAb engineering?

A

Changing glycans to modify antibody function
Sugars can be added by genetic engineering to express enzymes which add specific glycans
Sugars can be removed by knocking out genes which code for enzymes which are responsible for their addition

57
Q

How is antigen binding affinity an example of mAb engineering?

A

Antibodies can be isolated with strong affinities for an antigen
Site directed mutagenesis of the variable region can alter binding affinity to antigen
Usually higher affinity binding is required but sometimes may require antibodies with lower affinity e.g. to allow better penetration of a tumour

58
Q

How are Fc-mediated functions part of mAb engineering?

A

Choice of sub-class can influence the interaction with Fc receptors
Mutation in the amino acids in the mAb Fc region can be used to alter the interaction with Fc receptors and modulate functions such as ADCC and ADCP or half life
Changing the glycosylation of a mAb can alter structure and function e.g. in tests, de-fucosylated Abs have shown increased affinity for FcgRIIIa receptor and enhanced ADCC

59
Q

What are some examples of how mAbs are used?

A

In Enzyme-linked immunosorbent assays (ELISAs)
Western blotting
Flow cytometry

60
Q

How are mAbs used in medicine?

A

Used to diagnose/monitor diseases e.g. by ELISA or western blotting
Also used in therapy

61
Q

How are mAbs used in therapy?

A

Immunosuppression
Cancer
Infection
Autoimmune disease
Cardiovascular disease

62
Q

What is intravenous immunoglobulin (IVIG)?

A

A blood product purified from the serum of between 100-15000 people
Used to treat patients with antibody deficiencies

63
Q

Describe polyclonal antibody therapy for rabies

A

After a bite from a suspected rabid animal, polyclonal antibodies isolated from the serum of individuals who have been immunised with the rabies vaccine injected into the wound site - Human Rabies Immunoglobulin (HRIG)

64
Q

Describe polyclonal antibody therapy for tetanus

A

Tetanus immune globulin (TIG) is a plasma derived product obtained from donors immunised with tetanus toxoid vaccine
TIG contains Abs that provide temporary passive immunity to individuals who have low or no immunity to the toxin prod by Clostridium tetani as part of a post-exposure prophylaxis regimen following an injury in patients whose immunisation is incomplete or uncertain

65
Q

What is Palivizumab (Synagis)?

A

A humanised mAb used to treat respiratory syncitial virus (RSV)

66
Q

What is Raxibacumab (ABthrax)?

A

A human mAb for prophylaxis and treatment of inhaled anthrax (binds to toxin)

67
Q

What mAbs are used for COVD-19?

A

Sotrovimab
Casirivimab/imdevimab
Bamlanivimab/etesevimab

68
Q

Describe the mAbs used against COVID-19

A

All human mAbs
All bind to the spike protein
Use of 2 mAbs that bind to different epitopes of the spike protein reduces possibility of the product becoming ineffective due to mutation of virus

69
Q

What are some reasons to improve mAb therapy?

A

Efficacy - in cancer treatments, Abs rarely ever curative e.g. bevacizumab extended median survival by only 30% compared with standard chemotherapy
Side effects - e.g. first-infusion reactions; often fever-like symptoms; increasing potency of Ab or extending half-life in plasma might allow dose or frequency of administration to be reduced
Costs - high cost of development, Abs often required in high doses, mammalian cell prod is expensive so numerous other expression systems being explored

Infection and Immunity (33 decks)

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