L14. Immune-based Techniques in Research and Medicine 1

Question 1

Other than immune defence what are 2 other roles of antigen and why?

Answer 1

> Antibodies are widely used in research, as diagnostics and increasingly as a new class of therapeutic drugs.

> As bind to anything foreign or be designed to bind.

Question 2

What are 5 useful properties of antibody?

Answer 2

1. Diverse >10^8 specificities
   >Can make antibodies against any molecule
2. Specific, high affinity KD 10-8 – 10-9 M
   >Usually use IgG antibodies made in second immune response so have a high order of affinity
3. Domain structure stable, facilitates protein engineering
4. Multivalent improved binding, cross-linking can be useful
5. Effector properties useful in some techniques, therapeutics

Question 3

How were antibodies made for research?

Answer 3

> To make antibodies, immunise an animal and boost it to try generate secondary and memory responses against the injected protein.

> Producing antisera (serum containing antibodies to target antigen)

> Antibodies can be purified and labelled with a detectable (antibodies are colourless) tag e.g. a fluorescent probe, an enzyme or even gold particles.

Question 4

What is the most common role of antibodies in the lab?

Answer 4

ANTIBODIES ARE GENERALLY USED TO IDENTIFY AND LABEL MOLECULES IN COMPLEX MIXTURES

Question 5

What do antibodies recognise?

Answer 5

Antibodies recognise EPITOPES (shapes) on an ANTIGEN

Question 6

What are the 2 types of epitope antigen, and what happens if you boil them?

Answer 6

1. Linear epitope – adjacent in sequence (non-conformational) amino acids close on a peptide chain
   >If boiled the antigen, antibody will still recognise.
2. Discontinuous epitope – non-adjacent (conformational) amino acids which are distant on a peptide but when folded are close.
   >If boiled antigen, antibody won’t recognise.

Question 7

Why does a protein have several epitopes where an antigen can bind?

Answer 7

A protein is a large molecule so will have several epitopes on its surface where antibodies can bind.

Question 8

What are the 3 different ways antibodies can bind to antigen, which is stronger and what is the effect of these different ways?

Answer 8

1. Monovalent Binding to a Single Epitope: An antibody can bind monovalently, meaning one of its Fab arms binds to a single epitope on an antigen. This type of binding is generally weaker because only one arm of the antibody is engaged.
2. Divalent Binding to Repeated Epitopes on the Same Antigen: When an antigen has multiple identical epitopes, an antibody can bind bivalently to two separate epitopes on the same antigen molecule. This is a stronger interaction as both Fab arms of the antibody are engaged.
3. Cross-linking of multiple antigen molecules: An antibody can also cross-link two separate antigen molecules if each antigen has an epitope that matches the antibody’s specificity. In this case, one Fab arm binds an epitope on one antigen molecule, and the other Fab arm binds to an epitope on a different antigen molecule.

> Bivalent binding and cross-linking can enhance antibody functions, such as opsonization, neutralization, and the activation of the complement system. Cross-linking is also significant in processes like agglutination, where pathogens are clumped together, making them easier targets for phagocytic cells.

Question 9

What does Immunogenicity mean?

Answer 9

The ability of an antigen to induce an immune response

Question 10

What 5 factors determine the immunogenicity (ability of an antigen to induce an immune response) of an antigen and why?

Answer 10

1. Foreignness:
   >The immune system is more likely to respond to substances that are foreign to the body. Antigens that share a high degree of sequence homology with proteins in the recipient are less likely to be immunogenic because they are recognized as “self” by the immune system.
   >This is why animals like rabbits, sheep, and mice are often used to produce antibodies against human proteins, as their immune systems can recognize human proteins as foreign.
2. Molecular Size:
   >Larger molecules tend to be more immunogenic. Small molecules, typically those with a molecular weight of less than 1000 daltons, are often not immunogenic unless they are attached to a larger carrier molecule.
   >Carrier molecules increase the overall size and complexity of the antigen, enhancing its immunogenicity.
3. Chemical Composition and Structural Complexity:
   >Antigens with complex structures, including aromatic groups and charged residues, are typically more immunogenic.
   >Non-covalent interactions of antigen-antibody can be stronger with aromatic groups or charged residues.
4. Ability to provoke T cell responses
   >Effective immune responses, especially those involving IgG production, require T cell help.
   >T cells are more effectively activated by proteins than by small chemicals or carbohydrates. Thus, conjugating potential antigens to carrier proteins can enhance their immunogenicity by making them more recognizable to T cells.
5. Use of adjuvants – induce inflammation, “Danger signals”
   >Use adjuvant when immunising animal.

Question 11

What does conventional antisera contain and why?

Answer 11

> Conventional antisera contains polyclonal antibodies (product of several B cell clones and a
mixture of antibodies specific to different “epitopes” )

> Make antibody against an antigen but antigen will have multiple different epitopes where different B cells will recognise these, so the antiserum is the product of multiple B cell clones.
Produces polyclonal antibodies (as antisera contains multiple types of antibodies recognising different epitopes)

Question 12

What are the advantages and disadvantages of conventional antisera?

Answer 12

1. Advantages:
   >relatively cheap,
   >robust (may recognise partially denatured/unfolded antigen)
2. Disadvantages:
   >specific for multiple epitopes so may also recognise other proteins with this shared epitope(polyclonal antibodies)
   >need pure antigen to immunise,
   >can be difficult to standardise, as different animals respond differently to the same protein(each animal has varying immune response)

Question 13

Describe how conventional antisera can cause cross-selectivity and what issues this causes

Answer 13

As one antigen has many epitopes and some antigen may share epitopes with another antigen, so some antibodies may be specific to both (cross-react), this creates an issue when we want an antibody for a specific epitope (shape).

Question 14

What does a monoclonal antibody target and what are they derived from?

Answer 14

Specific for a single epitope, derived from single B lymphocytes (Kohler and Milstein 1975).

Question 15

What are the 1) advantages 2) disadvantages of monoclonal antibodies?

Answer 15

1. Advantages:
   >highly specific to an epitope
   >can be standardised as can be stored for when needed,
   >pure antigen not needed for immunisation as will be cloned after woulds anyway.
2. Disadvantages:
   >often conformation sensitive (so if antigen is slightly damaged will not recognise them).
   >expensive

Question 16

Why was a new method needed to create monoclonal B cells?

Answer 16

Can’t isolate B cells in culture as die, needed to immortalise B cells

Question 17

How are monoclonal antibodies made in 7 steps?

Answer 17

1. Took B cells from mouse, immortalised by fusing with immortal cell line myeloma (usually cancer derived)
2. Myeloma cell line was selected which lacked enzyme HGPRT and couldn’t secrete own antibodies
3. Fuse lymphocytes taken from immunised animal and fuse with myeloma cells
4. Get rid of unfused B cells as die in culture
5. Need to select against unfused myeloma cells in selective media containing HAT (blocking purine synthesis for only pathway unbound cells can do without enzyme) but fused cells would have gained HGPRT enzyme from lymphocyte so could survive
6. Let cells grow and form more hybridomas (hybrid cancer cells)
7. Can culture these cells forever and collect the monoclonal antibodies secreted.

Question 18

What has been a major use of monoclonal antibodies in research?

Answer 18

> A major use of monoclonal antibodies has been in defining cell surface molecules

> Can use monoclonal antibodies to identify cell types e.g. T cells (CD3) subpopulations e.g. CD4 and CD8 (All T cells express CD3/ Subpopulations express CD4 and CD8).

Question 19

What does CD stand for and why?

Answer 19

CD stands for cluster of differentiation, classification system to help clarify which monoclonal antibodies recognise which molecule

Question 20

Other than measuring surface proteins, how else can monoclonal antibodies help define B and T cells?

Answer 20

Can use monoclonal antibodies to measure what stage of differentiation

Question 21

What is a promising future for monoclonal antibodies?

Answer 21

As a magic bullet to treat cancer

Question 22

Why couldn’t monoclonal antibodies treat cancer in the past?

Answer 22

> We can only make rodent monoclonal antibodies, not human, so are recognised as foreign in humans so can’t treat cancer.

> Rodent antibodies induce immune responses in human patients e.g. HAMA (Human Anti Mouse Antibody), “serum sickness”

Question 23

What is serum sickness?

Answer 23

Serum sickness is a severe reaction to the foreign protein

Question 24

What are 2 key pieces of antibody engineering, and how were they made and what were the issues with them?

Answer 24

1. Antibody chimeras were made
   >Splice mouse V region genes and fuse to human C region genes.
   >mouse V regions still immunogenic, so caused some HAMA responses.

2, “Humanised “antibodies
>Hypervariable loops are most needed for binding so splice human framework region genes and mouse CDR region genes a.k.a CDR grafting, so only part of mouse antibody is the CDR loops.
>Facilitated by immunoglobulin domain structure allowing sustainability throughout tinkering.
>The antibodies may lose affinity/specificity as sometimes framework regions can be important. Time-consuming.

Question 25

What are the 2 Strategies for developing fully human antibodies and describe them

Answer 25

1. 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.
2. ANTIBODY GENE LIBRARIES
   >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.

Question 26

Describe how antibodies are made from gene libraries using phage display?

Answer 26

1. Isolation of Genetic Material:
   >Isolate mRNA from antibody-producing cells (which can be sourced from blood, lymphoid tissue, or bone marrow).
   >Isolate a population of genes encoding antibody variable regions, typically obtained as cDNA from naïve B cells.
   >Reverse Transcription and Amplification:
2. Reverse transcribe the mRNA to cDNA.
   >Amplify the variable region (Fab or Fv) of the antibody gene by PCR.
   Cloning and Expression:
3. Clone the Fab or Fv cDNA into bacteria or phage to create an antibody library.
   >The variable (V) genes are fused to a bacteriophage coat protein gene using a phagemid vector, which can be displayed on the surface of bacteriophages.
4. Screening and Selection (Panning):
   >Screen the antibody phage display library against a solid-phase antigen (panning process).
   >Phages with desired V regions that specifically bind to the antigen are selected. The antigen-coated surface is used to capture the phages that display antibodies with the desired specificity; unbound phages are washed away.

Question 27

How did Professor Greg Winter create a library of synthetic human antibody genes, and how did they increase affinity of these antibodies?

Answer 27

> Cloned all Human V region genes, gave a library which would bind to any antigen as is 109 members and which would bind to any antigen. Then to take it further induced more variability by randomising part of molecule corresponding to CDR region, then would take this library expressed on surfaces of phage and use antigen to select which phage is attached to the antibodies.

> At first will be weak affinity but CDRs can be further mutated randomly to improve specificity/affinity (like affinity maturation) as some mutations resulted in higher affinity binding. Doing this process several times keeps increasing affinity and specificity. (Mimic of AID enzyme causing mutations in CDR regions).

Question 28

What are the 3 main ways of generating fully human antibodies?

Answer 28

1. TRANSGENIC mice expressing human immunoglobulin genes.
2. ANTIBODY GENE LIBRARIES
3. SINGLE B CELL ANTIBODIES (Like phage display but isolate single lymphocyte from patient tissue)

Question 29

What is the use of taking single B cell antibodies from a human, and what is its and what is an advantage?

Answer 29

> Single antigen-specific B cells from patient blood or lymphoid tissues are isolated using e.g. fluorescent antigen and fluorescence activated cell sorting (FACS). Expressed antibody V genes are amplified and cloned.

>

	○ Useful for generating antibodies against emerging pathogens, when someone is infected against a new virus we can isolate their B cells and select antibodies.

Question 30

What 2 forms do camels make of antibodies?

Answer 30

> Normal 2 heavy and 2 light chains

> Antibodies made of 2 heavy chains, but with just nanobodies as vairable regions (1 domain)

Question 31

What is a nanobody?

Answer 31

> Nanobodies are apart of llama heavy chain antibodies (just made of 2 heavy chains), the variable regions bind antigen very well and this variable region is the nanbody

> Nanobodies correspond to a single immunoglobulin domain which bind antigen tightly

Question 32

What are 3 advantages of using nanobodies?

Answer 32

> Chemically very stable

> Easily expressed in bacteria, yeast as are just a single immunoglobulin domain.

> Humanisation feasible as is just one domain

Question 33

What are the 4 formats of monoclonal antibodies used for therapy and what method produces them?

Answer 33

1. Fully mouse
   >Made using immortalised B cell line (myeloma)
   >Full mouse antibody so causes serum sickness.
2. Antibody chimeras
   >Genetic engineering: Splice mouse V region genes and fuse to human C region genes.
   >Human constant regions with mice variable regions.
3. Humanized
   >Genetic engineering: Hypervariable loops are most needed for binding so splice human framework region genes and mouse CDR region genes a.k.a CDR grafting, so only part of mouse antibody is the CDR loops.
   >Human antibody other than mice hypervariable loops.
4. Fully human
   >TRANSGENIC mice expressing human immunoglobulin genes.
   >ANTIBODY GENE LIBRARIES (e.g. phage display)
   >Fully human antibody