Unit 2 - Antibody therapeutics

Question 1

What is antithymocyte globulin?

Answer 1

Purified gamma globulin (serum of rabbits immunised with human thymocytes)

Question 2

How does antithymocyte globulin work?

Answer 2

Contains antibodies that binds to

• CD2
• CD3
• CD4
• CD8
• CD11a
• CD18
• CD25
• CD28
• CD44
• CD45
• HLA class I
• HLA class II

Question 3

What effect do the antibodies in antithymocyte globulin have?

Answer 3

Antibodies deplete circulating lymphocytes by direct cytotoxicity
- complement (CMC)
- cell-mediated (ADCC)
Binding to cell surface molecules block lymphocyte function, leading to induction of apoptosis

Question 4

What are the side effects of antithymocyte globulin?

Answer 4

Fever and chills with or without hypotension (major side effect)
Possibly premedicate with corticosteroids, H-blocker
Serum sickness and glomerulonephritis
Anaphylaxis
Leukopenia and thrombocytopenia
Risk of infection
Antibody development
- 68% of patients developed anti-rabbit antibodies

Question 5

How are therapeutic proteins produced?

Answer 5

Polypeptides produced in microorganisms and cells

• humans
• animals

Question 6

Give some examples of therapeutic proteins?

Answer 6

• hormones
• cytokines
• enzymes
• fusion proteins
• antibodies

Question 7

What are the five classes of antibody?

Answer 7

IgG
IgA
IgM
IgD
IgE

Question 8

What is the structure of IgG?

Answer 8

Monomer

Question 9

What two essential functions do antibodies perform?

Answer 9

• antibodies bind to an epitope on an antigen with the arms of the Y. Each arm or monovalent antibody fragment (Fab 1) domain contains a binding site, making each antibody molecule at least bivalent
• the Fc domain of the Y imparts the antibody with biological effector functions such as natural killer cell activation, activation of the classical complement pathway and phagocytosis

Question 10

What is a monoclonal antibody?

Answer 10

One type of antibody, complementary to one type of antigen, made by one type of plasma cell

Question 11

What is a polyclonal antibody?

Answer 11

Antibodies from multiple B cells that bind to a single antigen with specificity for MULTIPLE epitopes on that antigen

Question 12

What is a hybridoma?

Answer 12

A mouse B-lymphocyte cell and a tumour cells fused together so that as it divides many of the same antibodies are produced

Question 13

What are the advantages of therapeutic antibodies?

Answer 13

• ability to bind with high specificity and affinity to a wide variety of molecules
• stable molecules, thus ideal molecules to be used as targeting reagents

Question 14

What are the disadvantages of therapeutic antibodies?

Answer 14

• the first monoclonal antibodies were murine molecules and were recognised as foreign when infected into patients, leading to their elimination by the patient’s immune system
• in order to be effective, antibodies often need to interact with certain elements of the immune system such as receptors displayed on effector cells or the complement cascade. Murine antibodies could not do this

Question 15

What is a chimeric antibody?

Answer 15

Fusion of murine variable domains, responsible for the binding activity, with human constant domains

• 70% human
• fully human Fc portion
• less immunogenic in humans
• interaction with human effector cells and the complement cascade

Question 16

What is a humanised antibody?

Answer 16

Hypervariable loops of a fully human antibody were replaced with the hypervariable loop of the murine antibody of interest

• humanised antibodies are 85-90% human
• less immunogenic than chimeric antibodies

Question 17

What technique relies on the ability to establish a physical link between a protein and gene encoding this protein?

Answer 17

In vitro selection - phage display

Question 18

What is displayed at the surface of the phage in phage display?

Answer 18

A protein fused to a filamentous phage capsid protein

Question 19

What are transgenic ‘humanised’ mice?

Answer 19

Mice with fully human antibodies

- created by replacing the entire mouse IgG repertoire with a human repertoire

Question 20

What do humanised mice produce on immunisation?

Answer 20

Human IgGs

- conventional hybridoma techniques can be used to clone human antibodies

Question 21

What is the advantage of using transgenic ‘humanised’ mice?

Answer 21

Directly lead to full length IgG, which is often the preferred format for therapy

Question 22

What are the disadvantages of using transgenic ‘humanised’ mice?

Answer 22

Cannot be used effectively when

• the immunogen is toxic
• the targeted antigen shares a high degree of homology with its murine ortholog

Question 23

What are the mechanisms of action of therapeutic proteins?

Answer 23

• blocking
• signalling
• targeting
• CDC (complement-dependent cytotoxicity)
• ADCC (antibody-dependent cell-mediated cytotoxicity)

Question 24

between Murine, chimeric, humanized and human which antibodies have the least potential for immunogenicity?

Answer 24

Human have the least potential to cause immunogenicity

Question 25

what are the limitations of antibody therapy?

Answer 25

The limitations of traditional antibody-based therapies include the high cost associated with production of complex biomolecules in mammalian cell culture systems. In addition, the size of the molecule, coupled with sensitivity to proteolytic degradation and the quantities of clinical grade material required to obtain a therapeutic effect, limits delivery routes to intravenous and subcutaneous delivery, and in so doing reduces the range of therapeutic indications that would warrant such approaches.