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Medical Biotechnology > Therapeutic Proteins L2-7 > Flashcards

Therapeutic Proteins L2-7 Flashcards

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USMLE® Step 1 flashcards
1
Q

Bacterial REs cut DNA at specific (_-_ bp) recognition sequences.

A

4-8

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2
Q

RE’s are used as a defensive measure by bacteria to cut up ____1____ genomes to stop them from ____2____.

A
  1. Viral
  2. Replicating
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3
Q

RE’s cut at specific recognition sequences, leaving what?

A

Sticky ends

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4
Q

After RE cutting, ____1____ joins the 2 DNA strands together. This requires the ______ 2 ______ and an energy source in the form of ATP.

A
  1. Ligase
  2. Sticky ends
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5
Q

Define a vector (genetics).

A

A carrier for genetic recombination.

or

A bacteriophage or plasmid which transfers genetic material into a cell, or from one bacterium to another.

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6
Q

We are able to use artificial chromosomes to control gene expression (turn on and off).

An example of this is the Lac operon.

Where would the Lac operon be inserted to control gene expression?

What turns it on and off?

A

Just before gene of interest.

The presence of the sugar lactose (on), absence of lactose (off). Also, in the presence of glucose, the lac operon will be turned off as glucose is a preferred source of energy.

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7
Q

Why can’t E.coli and other less complex organisms make all proteins that can be formed in humans?

A

They are unable to make many of the post-translational modifications that human cells can.

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8
Q
A
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9
Q

List the advantages of recombinant expression systems in prokaryotes. (4)

A
  • Ease of culture
  • Rapid cell growth
  • Expression easily induced
  • Easy retrieval and purification of product
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10
Q

List the disadvantages of recombinant expression systems in prokaryotes. (4)

A
  • Can have low expression levels
  • Rapid and severe product degradation
  • Missing post-translational modification
  • Inability to glycosylate
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11
Q

List the advantages of recombinant expression systems in simple eukaryotes. (4)

A
  • Established technology
  • High level of product mg/g of cells
  • Cost effective
  • Relatively easy to adapt for industrial scale production
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12
Q

List simple eukaryotes used in recombinant expression systems. (7)

A
  • Methylotropic yeasts (use methanol as energy source)
  • Candida boidinii
  • Hansenula polymorpha
  • Pichia pastoris
  • Saccharomyces cerevisae
  • Trichoderma (filamentous fungi)
  • Aspergillus (filamentous fungi)
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13
Q

Saccharomyces cerevisiae (bakers yeast) is a simple eukaryotic expression system that has been successfully used to make viral fragments for vaccines (HPV).

However, what is the major disadvantage of using it?

A

The addition of mannose rich branched chains at N glycosylation sites induces an immune response (seen as foreign as human cells do not use mannose for glycosylation).

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14
Q

______ is used to produce pectinase which helps release juice from fruits (E.g. useful for wine industries).

A

Aspergillus

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15
Q

Name a eukaryotic algae used as a recombinant expression system.

A

Chlamydomonas reinhardtii

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16
Q

Define baculovirus.

A

A viruses that infect insects.

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17
Q

List the advantages of recombinant expression systems in mammalian cell lines. (3)

A
  • Perform full set of post-translational modifications
  • Some cells have the ability to secrete the protein product into the culture media
  • Easier recovery and purification
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18
Q

List the disadvantages of recombinant expression systems in mammalian cell lines. (4)

A
  • Complex culture conditions required
  • Purification from the complex culture media may be required
  • Slow growth rate
  • Expensive
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19
Q

Why use malign cells in expression systems?

A

The cells are immortal, therefore meaning they will keep producing product indefinitely.

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20
Q
A
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21
Q

List the advantages of recombinant expression systems in transgenic plants. (3)

A
  • Easy to grow
  • Low cost
  • Easy to deliver as drug
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22
Q

List the disadvantages of recombinant expression systems in transgenic plants. (4)

A
  • Does it change the taste of the food?
  • How stable is it?
  • Personal choice
  • Where will the gene and protein get to
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23
Q

Read

Therapeutic proteins have revolutionized the treatment of many previously unmet medical needs.

Despite this there are still problems with

  1. Low activity
  2. Rapid clearance
  3. Immunogenicity
A
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24
Q

How many marketed therapeutic proteins are glycoproteins?

A

2/3

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25
Q

Half of human proteins are glycosylated with carbohydrate structures at one or more sites. Glycans are added by post-translational modification. The glycan component of a recombinant glycoprotein is very important as it affects what? (7)

A
  • Pharmacokinetics
  • Bioactivity
  • Secretion
  • In vivo clearance
  • Solubility
  • Recognition
  • Antigenicity
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26
Q

Define the quantitative aspects of glycosylation.

A

How much glycosylation.

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27
Q

Define the qualitative aspects of glycosylation.

A

Glycosylation occurs at which bonds, which sugars.

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28
Q

Define glycosylation.

A

Glycosylation is the process whereby oligosaccharides are added to the protein during synthesis and processing through the ER and Golgi apparatus.

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29
Q

Read

Glycoproteins occur as heterogeneous populations of molecules called glycoforms. The structure of a glycan is not governed by a predetermined template (as it is for proteins) and a variety of structures are possible for any one protein. The potential variability of glycoforms presents a very real difficulty to industrial production and for regulatory approval of therapeutic glycoproteins. The glycoforms produced will depend on which sugars were available at the time (bioavailability). This may depend on diet, stage of the cell cycle, any current immune response etc. (this is because these have an effect on the metabolism and therefore which sugars are produced).

A
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30
Q

Name and describe the 2 forms of glycosylation.

A
  • N-linked = sugars attach to the extra nitrogen group of the R chain
  • O-linked = requires either an oxygen or a hydroxyl group as one of the R chains on the protein. The amino acids that fit this stipulation are threonine and serine.
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31
Q

What is the most common sugar that attaches via N-glycosylation?

A

Mannose

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32
Q

Which sugar most commonly attaches as a side chain in glycosylation?

A

Fucose

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33
Q

Glycosylation

____1____ acid tends to attach to the ends of proteins. The end of the proteins are often the part that interacts with receptors so whether or not a protein has a ____1____ acid attached to it can have a profound effect on its function.

____1____ acid is ____2____ charged.

A
  1. Sialic
  2. Negatively
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34
Q

Read

Plants almost exclusively use arabinose for N-linked glycosylation.

Bacteria mainly use mannose.

This outlines how if we tried to produce a protein in plants and bacteria, though the amino acid sequence of the generated proteins would be the same, the differing glycosylation’s would cause them to act differently. Again, what has been produced are different glycoforms of the same protein.

A
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35
Q

GlcNAc and GalNAc often start off the _1_-glycosylated chains. These are really important sugars for helping the ____2____ happen.

A
  1. O
  2. Branching
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36
Q

Where do N-glycans bind?

A

Glycan is bound via a N-glycosidic bond to Asparagine within the consensus amino acid sequence (sequon) - Asn-X-Ser/Thr (where X can be any amino acid other than Pro or Asp).

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37
Q

N-glycans can be split into three groups.

List them.

A
  • High mannose type
  • Hybrid type
  • Complex type
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38
Q

All N-glycans have a common ______ core.

A

Pentasaccharide

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39
Q

High Mannose oligosaccharides have from _1_ to _2_ mannose residues.

Arranged as mono-, bi-, tri-, tetra- and penta-antennary structures.

A
  1. 3
  2. 60
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40
Q

Read

How do we produce N-glycans?

  1. Transcription in nucleus
  2. Moves to the RER (contains many ribosomes)
  3. Translation in these ribosomes
  4. Protein is formed on the inner membrane of the RER
  5. N-glycosylation occurs

The majority of the sugars we want are found in the cytoplasm.

In N-glycosylation, the glycan is generated separately and then attached to the protein. This is different to O-glycosylation.

N-glycan assembly is started by Dolichol phosphate, which is bound to the membrane of the ER. The phosphate groups (which give it activity) attract sugars which are then attached by enzymes in the cytosol. Once the core has been formed an enzyme will actually flip the membrane, internalizing the sugar core within the ER. Further modification of the core proceeds to make the sugar chains somewhat unique. As the ribosome finished producing the protein, the ribosome recognises the consensus sequence on the protein and using the energy from the phosphate groups attaches the glycan to the Asn on the protein, thus glycosylating the protein.

As the protein is being produced (and the formed part being thrust into the ER lumen), whenever a consensus sequence becomes available, the ribosome will attach the glycan. This helps the protein fold into its 3D structure due to the interference of the sugar molecules.

A
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41
Q

Read

N-glycans assembly

All N-linked glycans share the same core because they all come from the same precursor.

The precursor consists of a lipid (dolichol) linked to a glycan by a pyrophosphate bond.

The glycosylation is initiated in the ER where the N-glycan precursor is attached the the Asn-X-Ser/Thr consensus region by the enzyme oligosaccaryltransferase.

Series of glycosidases and glycosyltransferases in the Golgi.

Final products can be modified by the addition of sialic acid, poly-N-acetyl lactosamine or fucose.

The addition of sialic acid will give the glycoprotein its charge, allowing it to interact with various receptors.

A
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42
Q

Read

O-glycans

The oligosaccharides are attached to hydroxyl group of:-

  1. Serine or threonine – here the first sugar residue is normally N-acetylgalactosamine (GalNAc)
  2. Hydroxylysine (Hyl) –glycosylated by the attachment of a single Gal (Galactose) or glucosylgalactose disaccharide.
  3. Hydroxyproline (Hyp) – arabinose residue is linked.

Note that Hyl and Hyp only occur in collagens​.

This is why Vitamin C deficiency results in scurvy as the collagen falls apart and therefore so do the joints and fibres holding the teeth in.

A
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43
Q

In glycosylation, sugars are added on by ______.

A

Glycoltransferases

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44
Q

In glycosylation, sugars are removed by ______.

A

Glycases

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45
Q

In O-glycosylation there are 8 core sugar molecules that we know of.

These core groups are what determines the ______ groups.

A

Blood

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46
Q

O-linked glycosylation occurs exclusively in the ______.

A

Golgi

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47
Q

The first step for the assembly of the mucin type O-glycans is the addition of an N-acetylygalactosamine (____1____) residue to a Ser/Thr by a ____1____ ____2____.

A
  1. GalNAc
  2. Transferase
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48
Q
A
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49
Q

Read

Glycosylation is not under the direct control of the genome

Although the enzymes that synthesize individual monosaccharides and the glycosyltransferases that add these monosaccharides to glycan chains are defined by the genome, their activity in any given cell at any given time cannot be predicted.

It is affected by a multitude of factors, such as general cell metabolism and the rate of cell growth.

A
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50
Q

Read

Glycans play an important role in establishing or maintaining the integrity of glycoproteins. They do this by:

Biological Activity

  • ​Enhance thermal stability
  • Provide protection from proteases
  • Improve stability
  • Inhibit aggregation of proteins

Protein folding

  • Negatively charged glycans can interact with positively charged amino acid side chains

Receptor binding

  • Erythropoietin (EPO) is a sialoglycoprotein
  • Secreted by the mature kidneys in response to ↓pO2
  • Stimulates erythrocyte production from the blood marrow

Antibody effector function

  • Antibodies with low fucose enhance NK cell activity
  • Antibodies with high fucose content more effectively recruit polymorphonuclear cells

​Pharmacokinetics, clearance and half-life

  • In vivo potency of a drug is strongly associated with circulating residence time
A
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51
Q

Read

Glycans play an important role in establishing or maintaining the integrity of glycoproteins.

Follicle stimulating hormone (FSH) has a short half life. Long acting FSH can be created by added glycosylation. Adding mannose (bacteria) reduces half-life.

A
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52
Q

Read

Growth hormone (hGH)

  • Mixture of peptides that differ in their primary structure.
  • The principal form is a single-chain 191aa non-glycosylated protein of 22kDa

By late 1980s large quantities of recombinant rhGH allowed therapeutic uses in :

  • Children with hGH deficiency
  • Enhances post-surgical repair in adults
  • Slows wasting in AIDS and a number of other conditions
  • Used by athletes (but is banned)
A
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53
Q

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Insulin

  • Originally derived from the pancreas of pigs and cows
  • But is slightly different causing inflammatory responses at injection sites and neutralisation of action
  • Recombinant DNA technology produces human recombinant insulin in E coli
  • There are many different forms of human recombinant insulin which have different characteristics. Some may exhibit a rapid effect whilst others have an extended effect
A
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54
Q

Deficiency of Factor VIII = ______ _

A

Haemophilia A

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55
Q

Deficiency of Factor IX = ______ _

A

Haemophilia B

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56
Q

Read

Interferons

  • Group of small proteins (140-170 amino acids)
  • Antiviral agents and retard the growth of tumour cells
  • Divided into three main types:

  1. B lymphocytes (IFN-a)
  2. Fibroblasts (IFN-b)
  3. T-lymphocytes (IFN-g)
A
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57
Q

Describe the 3 main ways in which interferon-α functions.

What is interferon-α an effective treatment for? (4)

A
  • Directly affects cancer cells by interfering with cell growth and multiplication
  • By increasing the degree to which cancer cells present antigen on their surface
  • Activating NK cells

Effective in renal cell carcinoma, malignant melanoma, multiple myeloma and some types of leukaemia

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58
Q

A secreted B cell receptor = ?

A

Antibody

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59
Q

What type of bonds hold the Fc and Fab fragments of an antibody together?

A

Disulphide bonds

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60
Q

State the two basic functions of antibodies.

A
  • Bind specifically to regions (epitopes) on pathogen/Ag that elicited the immune response.
  • Recruit cells and molecules to destroy the pathogen/antigen once the antibody is bound to it.
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61
Q

The antigen-binding region of the antibody is known as the ______ region.

______ region interacts with pathogen/antigen.

A

Variable

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62
Q

The region of the antibody involved in the effector functions is known as the ______ region.

______ chain interacts with host cells.

A

Constant

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63
Q

Define epitope.

A

The part of an antigen molecule to which an antibody attaches itself.

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64
Q

Antibodies are bivalent. In this context, what does this mean?

A

Antibodies have two identical antigen binding sites.

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65
Q

Constant heavy region attached to VH (Fab CH) is the region of the antibody that interacts with the ______ pathway.

A

Complement

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66
Q

Both heavy and light chain polypeptides are constructed from individual, equal sized (110 aa, 12.5 kDa) domains.

Therefore an antibody weighs how much?

A

150 kDa

67
Q

Antibodies are very soluble. This is one of the key features of an antibody as it needs to be able to stay in solution in high concentrations in our ______.

A

Serum

68
Q

______ cuts disulphide bonds between Fab fragments and Fc fragments.

A

Papain

69
Q

Define affinity and avidity.

Whats the difference?

A

Affinity measures the strength of interaction between an epitope and an antibody’s antigen binding site.

Avidity gives a measure of the overall strength of an antibody-antigen complex.

Therefore avidity is the cumulative strength of all affinities.

70
Q

______ chops up Fc region leaving a Fab2 fragment.

A

Pepsin

71
Q

Read

In the human genome (maybe antibody specific parts) there are too few genes to satisfy the variability needed for the many variable regions of the many antibodies.

So the antibodies are much more variable than their genes allow them to be.

How?

Graphs have shown the comparison of average variability of variable regions of numerous antibodies.

From this we discovered that that variable regions are made up of multiple FRs (Framework regions) and HVs (Hyper-variable regions).

  • FRs are still variable but mainly provide the framework upon which the HV sit within
  • HVs (also called CDRs, complementary determining regions)

This is very important to understand to be able to design our own antibodies.

A
72
Q

Serum from an immunized person/animal is known as ______.

A

Antiserum

73
Q

Antiserum will contain antibodies that bind the Ag (as well as those that were present before immunization), along with other ____1____ blood components (growth factors, other proteins etc.) but does not contain ____2____ or clotting proteins.

A
  1. Soluble
  2. Cells
74
Q

An antiserum may contain many different antibodies that bind the same Ag (but different epitopes).

What does this allow?

A

Greater antibody binding on a pathogen as they do not all compete for the same site.

75
Q

How can we purify antibodies from antiserum?

A

All antibodies are 150 kDa. Therefore by using chromatography we can separate out all molecules at this weight.

76
Q

What is the main limitation of using immunized antisera as a technique for antibody production?

A

Once the antiserum has been used, then another individual will need immunizing and the antibodies generated will never be exactly the same.

77
Q

Define monoclonal antibody.

What is the benefit of using monoclonal technology over antisera?

A

An antibody that is being made by a cloned B cell.

The antibodies produced are identical.

78
Q

Read

Monoclonal antibody technology

What if the individual B cell clones could be kept in culture to continuously produce a single antibody.

Patients with multiple myelomas (immortal cancer cells) were known to produce large amounts of homogenous antibodies, although the specificity of the antibodies were unknown.

Georges Kohler, Cesar Milstein and Niles Jerne devised a method for fusing mouse myeloma cells with B cells that were making antibodies of known specificity.

A
79
Q

Read

Monoclonal antibody technology

Immunization protocols

  • A conventional immunization protocol can be followed using a range of administration routes/adjuvants and animals may be kept for up to one year before B cell isolation
  • Animals must be boosted (preferably intravenously) 4 days prior to the fusion
  • This ensures that the relevant lymphocytes are at their peak of proliferation rather than antibody secretion
  • The source of lymphocytes is normally the spleen, although lymph nodes may be used.
A
80
Q

Monoclonal antibody technology

When the B cells became myelomas they stopped producing antibodies. Therefore they will not produce antibodies that will interfere with the production of the antibody we want but they are also still B cells in origin therefore making ______ viable.

A

Fusion

81
Q

Monoclonal antibody technology

All cells need to make nucleotides to make their DNA.

Aminopterin = Inhibits cells from making the nucleotides through the De Novo pathway. This kills the cells.

Nucleotides can be reused though via the salvage pathway. To do this cells must have TK (Tyrosine kinase) and HGPRT to make pyrimidines and purines respectively.

So if Aminopterin is added, cells can still survive if they have these enzymes.

Kohler and Milstein managed to find myelomas that had lost HGPRT expression.

Therefore adding Aminopterin to these myelomas would kill them.

This means that only the fused cells would contain HGPRT and therefore would survive.

The B cells will naturally die over time as they are not immortal.

Thus the hybridomas are selected for by HAT.

HAT = Hypoxanthine, Aminopterin, Thymidine

A
82
Q
A
83
Q

Read

Antibody screening tests for HAT resistant hybrids

Approximately 2 weeks after the fusion it will be necessary to determine which of the mini hybrid cultures are producing the desired antibody. The cultures will be tested by a screening test. Normally, the hybrids will be tested for antibody production (ELISA). Hybrids that produce antibody of interest will then be re-tested to characterize their specificity even further.

Cloning

Each HAT resistant hybrid will have originated from a single myeloma cell fusing to a single B cell clone. It’s likely that more than one hybrid exists each producing an antibody of interest. Therefore the mixture of hybrids is counted and diluted in a series of steps such that the theoretical number of cells per 100 ml (volume added to each well in a 96 well plate) is 0.3 (0.3 cells per well statistically ensures that when a well has a cell within it, it will only have 1 cell – thus separated). These cultures are normally supplemented with feeder cells (peritoneal macrophages, splenocytes) which secrete growth factors. Within two weeks of cloning the supernatant can be re-tested for antibody.

A
84
Q

Read

Polyclonal antibodies have been used as therapies for many years.

Due to their vast diversity and specificity monoclonal antibodies have the potential to be used in the therapy of a large number of diseases.

However most patients treated with mouse monoclonal antibodies will develop anti-mouse antibodies since they are only about 60-70 % similar to human antibodies.

This response is known as the human anti-mouse antibody (HAMA) response.

Some patients develop HAMA after a single dose of antibody whilst others require several doses but it normally occurs 10-30 days after administration.

Further doses of the monoclonal antibody treatment lead to the production of immune complexes and rapid clearance to the liver and spleen. Thus it’s likely the drug (antibody) will not reach its intended target.

In addition at least 10% of the population have naturally occurring anti-mouse antibodies.

Would HAMA response be reduced or stopped by replacing the non-Ag-specific regions of mouse antibodys with those from human antibodys i.e. “humanizing” mouse antibodies?

The protein domain structure of antibodies is ideal for this purpose as each domain is encoded by a different genetic exon.

Genetic manipulation can therefore be used to graft different exons together resulting in CHIMERIC antibodies.

In addition the human constant regions with the most appropriate effector functions may be chosen.

A
85
Q
A
86
Q

Chimeric antibodies

Chimeric antibodies were first made by isolating and sequencing the genes coding for variable regions of the heavy and light chains from a mouse ____1____.

Mouse V region genes were inserted into separate expression vectors containing human heavy or light chain C region genes derived from human ____2____.

By ____3____ of these vectors into mammalian cell lines, chimeric antibodies can be expressed yielding up to 0.7 g/litre of culture medium.

As long as a cell has the ability to form ____4____ bonds, we can use it to produce chimeric antibodies – naturally only happens in B-cells.

A
  1. Hybridoma
  2. Myelomas
  3. Co-transfection
  4. Disulphide
87
Q

Give an example of a chimeric antibody + its target.

What is it used to treat?

A

Infliximab (anti-TNFa).

Used in the treatment of inflammatory diseases (particularly rheumatoid arthritis).

88
Q

Humanized rodent antibodies

Whilst many chimeric antibodies demonstrated reduced immunogenicity and were used with some success in many cases there still remained the problem of patient reactivity to the rodent _1_ regions.

Thus the next development was to reduce the rodent part of the antibody even further by grafting only the ____2____ regions into the human framework

A
  1. V
  2. Hypervariable
89
Q

Technically, only the ______ region is needed to bind the antigen.

A

Hypervariable

90
Q

In superhumanised chimeric antibodies, which parts of the original non-human antibody are left?

A

Only the key amino-acids of the hypervariable region used in binding the epitope.

91
Q

Complementary determining regions (CDR) = ?

A

Hypervariable regions

92
Q

CDR grafting

Grafting the CDR regions from rodent monoclonals into a human antibody results in the transfer of specific antigen binding with ____1____ antigenicity.

Antigen binding by antibodies is not only dependent on the amino acid sequence of the CDR loops but also on their ____2____ which is determined by the framework regions of the variable region.

The framework regions thus provides the correct __3__ platform for the CDR loops, and even one amino acid change can significantly change the CDR loops’ binding properties.

Information from ____4____ studies and molecular modelling has allowed the design of optimal human frameworks for CDR-based Ag binding.

A
  1. Reduced/decreased/less
  2. Shape
  3. 3D
  4. Crystallographic
93
Q

Give an example of a superhumanised chimeric antibody + its target.

What is it used to treat?

A

Xolair/Omalizumab (human IgE).

Severe allergic asthma.

94
Q

The minimal fragment that still contains the whole antigen binding site is a heterodimer composed of the VH and VL.

This is called an ____ fragment.

If the VH and VL genes are artificially joined with a linker a single chain (sc)____ can be made

A

Fv

95
Q

An Fv fragment won’t have an effector function (does not contain the Fc regions that bind FcRs on human cells). Why may this not matter?

A

If therapeutically we just want to use them to block something and stop that something from having an effect on the body - as opposed to the usual antibody effector function.

96
Q

The CL and CH of the Fab fragment is only needed because of the ____1____ bond which holds the variable region together. But if we find another way of holding the V region together then we only need VL and VH. This is what has been done for __2__ fragments.

A
  1. Disulphide
  2. Fv
97
Q

In the scFv fragment the VH and VL domains are engineered so that they are joined by a hydrophilic, flexible, ____1____ linker which improves expression and ____2____ efficiency.

This is an alternative for the disulphide bond.

A
  1. Peptide
  2. Folding
98
Q

Name the most commonly used scFv peptide linker.

A

(Gly4Ser)3

99
Q

After dissociation of scFv into monomeric scFvs can be complexed into dimers (diabodies), trimers (triabodies) or larger structures.

The simplest designs are diabodies that have two functional antigen binding domains that can have the same specificty (bivalent diabodies).

Multiple scFvs increases the avidity, why?

A

Multiple affinities are being added together.

100
Q

Bispecific antibodies can recognise 2 epitopes, trispecific 3, etc.

These multimeric antibodies could be used to bring antigens together.

These constructs can be made by cell fusion methods, genetic manipulation (most common today) or chemical recombination.

A
101
Q

Name the modern method used to engineer multimeric antibodies.

A

PCR

102
Q

Read

Bi-specific antibody applications

  1. Of particular use in therapeutic applications are constructs of different Fab specificities where one binds to the target epitope on a tumour cell and the other binds to an effector T cell.
  2. bsAb may simultaneously block several receptors on a cell surface. E.g. A diabody that binds to both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor has been shown to inhibit the tumour growth.
A
103
Q

Why may trispecific antibodies be even more effective than bi-specific antibodies?

A

Due to tighter binding of effector cell or toxin to the target and enhanced effector function.

104
Q

Read

Future of BsAb

DART (dual affinity re targeting platform): capable of targeting many different epitopes with a single molecule.

Modular antibody technology: Allows small antibody fragments with full antibody functionality (Fcab).

Bicycle technology: Mini antibodies with two binding loops covalently attached to organochemical cores

Dual targeting domain antibodies: binds two targets and can be made in dimer, Fab-like or IgG like formats.

A
105
Q

What is the benefit of reducing the size of antibodies? (2)

A

It reduces the amount of regions that the immune system can recognise. Therefore is less likely to evoke an immune response.

It also becomes cheaper to make (less to build).

106
Q

What is the drawback of reducing the size of antibodies?

A

They are difficult to make as their solubility decreases.

107
Q

In 1993 it was discovered that Camelidae (camels, dromedaries and llamas) have a novel class of ______ antibodies that do not have light chains. More recently it has been shown that sharks have similar heavy chain antibodies (hcAbs).

A

IgG

108
Q

Heavy chain antibodies

hcAb have a molecular weight of about _1_ kDa.

The antigen binding domain consists of only a single heavy chain variable domain designated VHH in camel hcAb and VNAR in shark hcAb (to distinguish them from VHs).

Since the first constant domain is missing the VHH is immediately followed by the ____2____ region and CH2 and CH3 domain in camels and in shark hcAb the VNAR is followed by the ____2____ and 5 CHNAR.

A
  1. 95
  2. Hinge
109
Q

Read

Applications of hcAb

In vivo cancer imaging and cancer immunotherapy, anti-EGFR, from immune camel and llama.

Cytokine neutralization, anti-TNFα, from immune llama and alpaca.

Competitive enzyme inhibition – using serum from immune dromedary

Blocking virus secretion, Hepatitis B from Immune llama

Trypanosome drug delivery, variable surface glycoprotein, immune dromedary

Malaria diagnosis, AMA1, Immune shark

A
110
Q

Read

“Camelisation” of antibodies

To improve the solubility and to prevent non-specific binding residues located on the VL side of human VHs are replaced by VHH - like residues.

These mimic the more soluble VHH fragments.

This process is termed camelisation and these camelised VH fragments, based on the human framework, are expected to have significant advantages for therapeutic purposes in humans.

A
111
Q

Read

Plant antibodies

Transgenic plants have been used for the production of antibodies against dental caries, rheumatoid arthritis, cholera, E. coli diarrhoea , malaria, certain cancers, Norwalk virus, HIV, rhinovirus, influenza, hepatitis B and herpes simplex.

The most advanced is CaroRX, (anti-S. coccus IgA) produced in tobacco. This antibody prevents dental caries and has been subjected to phase II clinical trials in humans with favourable results. The mouth is first washed with an oral antiseptic to remove all bacteria and this is immediately followed by the application of CaroRX several times over a 2 week period.

A
112
Q

Define intrabody (intracellular antibody).

A

An antibody fragment that is expressed within a designated intracellular compartment.

113
Q

What do intrabodies cause?

A

The interruption or modification of the biological functions of a target protein.

114
Q

Describe the 5 ways intrabodies can be used in.

A
  • Sequestration of target protein from its normal subcellular compartment of action
  • Mediating enzyme function through blocking of the active site or modulation of its conformation
  • Disruption of signal transduction pathways
  • Induction of cell death by apoptosis
  • Selective degradation by the ubiquitin – proteosome pathway
115
Q

How are intrabodies constructed?

A

By cloning V regions of human (or mouse) antibodies into a range of viral vectors.

116
Q

Read

Intrabodies have shown potential in the treatment of:

  1. Recombinant Intrabodies as Molecular Tools and Potential Therapeutics for Huntington’s Disease
  2. Cancer – to alter neoplastic properties of cells (including knockdown of growth-factor receptors, angiogenesis related receptors etc.
  3. HIV – interruption of the HIV-1 lifecycle.
A
117
Q

Read

We can transduce cells in a person with a virus which will encode an antibody which will enter the cells in that person.

Not only must we get the antibody into the cell but we must also ensure it reaches its target. We can do this through intracellular targeting.

  • Can insert small base sequences into proteins to localise them to specific places within the cell.

The antibodies we design must be suitable to the area we want them to work. Environmental factors like pH are different throughout the cell and thus the antibody must be suitable for where it will be localised.

A
118
Q

Define bacteriophage.

A

Viruses that infect bacteria.

119
Q

If you immunize an animal it will produce a huge number of antibodies against the vaccine. We may want to extract specific antibodies and clone them for mass use.

How though, do we target and extract the correct antibodies?

Well first of all the antibodies produced are in too small numbers to be individually detected. So we need to amplify up all antibodies generated. Scientists did this by use of ______.

A

Bacteriophages

120
Q

Phage Display

Just like other viruses, bacteriophages can be used as ____1____ for genetic modification applications.

Phage display differs from other expression systems in that the gene of interest is spliced into the gene sequence of one of the phage’s coat proteins so that the foreign amino acid sequence is genetically fused to endogenous amino acids of the coat protein to make a ____2____ protein.

This ____2____ coat protein is incorporated into phage particles and released in high amounts from the infected bacteria.

This allows us to fuse antibodies to the coats of these phages. They then rapidly ____3____ to generate incredible amounts of the antibody. This is a far more efficient way of mass producing the antibodies than creating and cloning B cell hybridomas.

A
  1. Vectors
  2. Hybrid
  3. Divide/replicate
121
Q

What is a phage display library?

A

A very large heterogeneous mixture of individual phage clones, each carrying a different DNA insert and therefore displaying a different peptide on its surface.

122
Q

Phage display library

Each peptide in the library can replicate, since when the phage to which it is attached infects a fresh ____1____ cell, it multiplies to produce a huge crop of identical progeny phages displaying the same peptide.

The antibodies are detectable because the phage will present the antibody on its ____2____.

A
  1. Bacterial
  2. Surface
123
Q

What are filamentous phages?

A

Flexible rods about 1mm long by 6nm in diameter. They are composed mainly of a tube of helically arranged molecules of the 50-residue major coat protein pVIII.

124
Q

E.coli (and most gram –ve bacteria) are covered with pili that are protein tubes involved in ____1____. Some E. coli have a long conjugation pili on the surface (F or sex pili) and these can pass ____2____ from a donor (male) to a recipient (female) bacterium.

A
  1. Attachment
  2. DNA
125
Q

Read

Peptide display on filamentous phage

Filamentous phages are flexible rods about 1mm long by 6 nm in diameter. They are composed mainly (87% by mass) of a tube of helically arranged molecules of the 50-residue major coat protein pVIII. There are 2700 copies in wild type virions.

Inside this tube lies the single standed viral DNA (ssDNA; 6407-8 nucleotides in wild type phage).

At one tip of the particle there are 5 copies each of the minor coat proteins pIII and pVI. Minor coat proteins pVII and pIX are found at the other tip.

The phage infect the E. coli by attachment of the N-terminal domain of pIII to the tip of the pilus.

If an antibody gene is cloned into a phage vector as a fusion with the pIII gene then the antibody is expressed or “displayed” on the surface of the phage.

This enables the phage expressing the desired antibody specificity to be selected from a mixture by binding to its Ag and then eluting off the bound phage.

This process is known as panning and is repeated several times enriching the original random mixture several thousand fold at each round.

The phage containing genes for the specific antibody can then be reinfected into bacteria and amplified.

These phage display antibodies can be used directly in techniques such as ELISA or soluble antibody fragments can be easily generated by genetic manipulation.

If the antibody is needed for therapeutic purposes it may be necessary to tag on a constant region from a cloned Fc library and then the whole antibody construct can be expressed in a eukaryotic cell system.

A
126
Q
A
127
Q

Define ribosome display.

A

A cell free system for the in vitro selection of proteins and peptides from large libraries.

128
Q

Read

Ribosomal Display

Ribosomal Display is a cell free system for the in vitro selection of proteins and peptides from large libraries. It uses the principle of coupling individual proteins (phenotype) to their mRNA (genotype), through the formation of stable protein-ribosome-mRNA (PRM) complexes. This allows the simultaneous isolation of functional protein and encoding mRNA which is then converted and amplified as DNA.

The key feature of ribosome display is the generation of stable protein-ribosome-mRNA complexes (PRM) complexes via ribosome stalling. Thus the protein and mRNA remain associated.

A
129
Q

Define in-vitro translation.

A

Ribosomes purified from cells can be given RNA and they will produce protein from this.

130
Q

Describe the 2 strategies of ribosomal display.

A
  • Addition of antibiotics such as rifampicin and chloramphenicol (prokaryotes) or cyclohexamide (eukaryotes) to halt translation.
  • Deletion of the stop codon normally recognised by release factors which trigger detachment of the polypeptide from the mRNA.
131
Q
A
132
Q

Prokaryotic Ribosomal Display

The first description of ribosomal display was for peptide selection from a random ____1____ library expressed in E. coli which was used to generate polysome complexes. ____2____ was stopped using chloramphenicol and specific complexes were captured with immobilised ____3____ bound on microtitre wells.

The trapped polysomes were disrupted with EDTA to release bound ____4____ which was then converted and amplified to cDNA by PCR.

The modified prokaryotic method generates PRM complexes through deletion of the 3’ terminal stop codon from ____1____.

A
  1. DNA
  2. Translation
  3. Antibody
  4. mRNA
133
Q
A
134
Q

Eukaryotic Ribosomal Display

A eukaryotic ribosome display system –ARM (antibody-ribosome-mRNA) has been developed for the selection of scFV fragments in a ____1____ free system.

____2____ of the stop codon is used to generate eukaryotic PRM complexes in a simple and rapid procedure

The ARM complexes are captured on antigen coated magnetic ____3____.

DNA may be recovered from the trapped mRNA without dissociation of the ribosome complex.

A
  1. Cell
  2. Deletion
  3. Beads
135
Q

ARM (antibody-ribosome-mRNA) display

A
136
Q

Transgenic mice expressing human IgG can also be modified to produce human antibodies.

Can effectively ____1____ a monoclonal antibody in a mouse. The mouse and its progeny will keep making this antibody.

Transgenic mice expressing a monoclonal antibody acts as a ____2____ cell in that it only produces one antibody – all other antibody genes are turned ____3____.

A
  1. Immortalise
  2. B
  3. Off
137
Q

Give 3 examples of monoclonal antibody therapeutics synthesised in genetically engineered mice.

A
  • Medarex
  • Abgenix
  • Regernon
138
Q

Human Antibody Techniques

A
139
Q

How do antibodies kill cells?

A

Via Antibody-dependent cell-mediated cytotoxicity (ADCC).

140
Q

Define Antibody-dependent cell-mediated cytotoxicity (ADCC).

A

A mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies. In humans, ADCC is usually mediated by IgG.

141
Q

Antibodies as therapeutics

____1____ (anti-CD3): Immunosuppressant (prevents acute rejection)

____2____ (anti-CD20): non-Hodgkins lymphoma

____3____ (anti-CD20): B-cell follicular lymphoma (Note some fatal side effects)

____4____ (anti-CD20): non-Hodgkins lymphoma and rheumatoid arthritis

____5____ (Infliximab): anti-TNFa Rheumatoid arthritis, Chron’s disease, Psoriatic arthritis, Ulcerative colitis, Ankylosing spondylitis

____6____ (anti-glycoprotein (Gp)IIb/IIIa): blocks platelet aggregationa and prevents cardiac ischemic complications

____7____ (anti-alpha chain of IL2 receptor): prevents organ rejection through inhibition of T cell proliferation by binding to and blocking IL-2 receptor

____8____ (anti-Epidermal growth factor receptor (EGFR)): Treats large bowel cancer (colon and rectum). Experimental for lung cancer and head and neck cancer)

____9____ (anti-respiratory syncitial virus (RSV)): Protects against infection with RSV

____10____ (anti-HER2): Breast cancer

____11____ (anti-CD25): Immunosuppresive (organ transplants)

____12____ (anti-CD33): Acute myeloid leukemia (delivers toxic drug to leukemic cells (ozogamacin))

____13____ (anti-CD52): B cell chronic lymphocytic leukemia and Multiple sclerosis (experimental?) (CD52 is found on T and B lymphocytes)

____14____ (anti-IgE): Corticosteroid resistant asthma (other allergies?)

____15____ (anti-CD11a): Immunosuppresive. Treats psoriasis, juvenile rheumatoid arthritis (one of two components, with CD18, of lymphocyte function associated antigen 1 – LFA-1 which plays a role in cell adhesion)

A
  1. OKT3
  2. BEXXAR
  3. Zevalin
  4. Rituxan
  5. Remicade
  6. Reopro
  7. Simulect
  8. Erbitux
  9. Synagis
  10. Herceptin
  11. Zanapax
  12. Mylotarg
  13. Campath
  14. Xolair
  15. Raptiva
142
Q

Read

Antibody Nomenclature

  • Antibodies have a scientific name and a trade name. The scientific name is made of 4 syllables.
  • The first is the unique name
  • The second is the general target e.g. li or lim = immune
  • The third is the source, mo = mouse, xi = chimeric, zu = humanised, u = human
  • The last is mab

E.g. 1

Remicade is the trade name for infliximab

  1. Inf – name
  2. Li – immune system target
  3. Xi – chimeric
  4. Mab – antibody

E.g. 2

Rituxan is the trade name for rituximab

  1. Ri – name
  2. Tu – tumour
  3. Xi – chimeric
  4. Mab - antibody
A
143
Q

Identify disease:

  • An inflammatory disease of the joints
  • Affects 1-2% of population
A

Rheumatoid Arthritis

144
Q

Read

One of the earliest successful application of monoclonal antibody therapy involves rheumatoid arthritis (RA).

There is still much of the underlying aetiology of RA which is unknown – infection, autoimmunity, synovial fibroblast transformation?

Whatever the cause there is a dysregulation of pro-inflammatory cytokine production including TNF a and IL-1 leading to joint destruction.

Diseases like RA are very heterogeneous – means that sufferers probably suffer from varying types of RA.

In inflammation cytokines appear very quickly and control many subsequent steps in immunology. As well as controlling this, cytokines such as TNFa also control how bones and cartilage are remodelled. Experiments showed that all of the enzymes that cause the breakdown of bone and cartilage are controlled by TNFa. So not only do these cytokines induce inflammation but they also cause this breakdown which leads to the symptoms of RA and other similar diseases.

A
145
Q

Although many ____1____ are involved in RA it has been shown that TNFa is at the apex of the cascade.

TNFa is directly involved in the generation of osteoclasts – activation of which leads to ____2____ loss.

TNFa is directly involved in the proliferation of synoviocytes – which express other mediators in the cascade.

TNFa is directly involved in the expression of collagenases by chondrocytes – leading to ____3____ destruction.

In short term cultures of rheumatoid synovial membranes anti-TNFa mAb not only inhibit the production of TNF-a itself but also inhibits other pro-inflammatory ____1____ e.g. IL-1, IL-6, IL-8 and GM-CSF.

This finding was reproduced in murine-collagen-induced arthritis.

Thus these in vivo and in vitro studies suggested that TNFa may be rate limiting in disease progression.

A
  1. Cytokines
  2. Bone
  3. Joint
146
Q

Diagram shows all of the many pathways controlled by TNFa and therefore the effect that blocking it has.

A
147
Q

Explain how Etanercept functions to treat TNFa.

A

Etanercept is a false receptor that is added and binds to TNFa thereby preventing it from binding the actual effector TNFa receptors.

148
Q

Use of Mabs in Transplantation

Rejection of transplanted organs is still a major barrier to transplantation success. Recognition of donor ____1____ molecules by the recipient as foreign initiates rejection of the graft.

There are three types of rejection:

Hyperacute rejection: occurs within ____2____ hours of the transplant and is due to pre-existing host antibodies to the graft.

Acute Rejection: occurs about a ____3____ after the transplant. T and B cells expand. T cells cause the graft cells to lyse and/or secrete inflammatory cytokines. The risk of acute rejection is highest in the first 3 months after transplantation. The onset of acute rejection is combated by treatment with the monoclonal antibody OKT3 (Muromonab CD-3)

Chronic Rejection: occurs months to years after the transplant and the process results in ____4____ leading to cell death.

Acute rejection can be treated, thus chronic rejection is the major cause of graft loss.

A
  1. MHC
  2. 24
  3. Week
  4. Fibrosis
149
Q

Use of Mabs in Transplantation

OKT3

A mouse monoclonal used to prevent acute rejection. OKT3 acts in two phases:

  1. Immediately after injection, circulating T cells are ____1____ as a result of opsinization and cytolysis.
  2. Antigenic modulation (CD3 complex is removed from the cell surface producing immuno-incompetent ____2____ cells.

OKT3 has been used since 1986 in renal transplants and has also been used for the treatment of acute rejection in liver and heart transplants.

A
  1. Depleted/reduced/decreased
  2. T
150
Q

Herceptin (Trastuzumab) targets HER-2 (Human Epidermal Growth Factor Receptor 2) which is expressed at high levels in 25% of ____1____ cancers.

Herceptin prevents transmission of the growth signal to the nucleus and stimulates ____2____ (where immune cells destroy cancer cells).

A
  1. Breast
  2. ADCC
151
Q

The National Institute for Health and Care Excellence (NICE) have recommended what for Herceptin therapy? (2)

A
  • Herceptin is given at three week intervals for 1 year or until disease recurrence (whichever is the shorter) as a treatment for early stage HER-2 positive breast cancer following surgery, chemotherapy and/or radiotherapy.
  • Cardiac function should be assessed before starting therapy and should not be offered to women with certain cardiac irregularities
152
Q

CD20 is only expressed on ______ cells.

A

B

153
Q

Gemtuzimab-ozogamacin (Mylotarg) targets CD33 which is expressed in 80% acute myeloid leukemia cases but not in normal ____1____ cells.

The antibody is linked to a cytotoxic agent and termed ozogamacin. It was approved in the UK in 2000.

The antibody binds to CD33 on leukemic cells. The antibody is internalized and then the cytotoxic agent is binds to DNA resulting in strand breaks and ____2____.

CD33 is also expressed on some healthy cells so these will also be affected.

A
  1. Haematopoieitic
  2. Apoptosis
154
Q

Alemtuzumab (Campath) targets CD52 which is expressed on B cells in patients with B cell chronic lymphocytic ____1____.

Humanized licensed in UK in 2001.

Acts by antibody dependant cellular cytotoxicity (ADCC) and ____2____ dependant cytotoxicity.

A
  1. Lymphoma
  2. Complement
155
Q

Ibritumomab tiuxetetan (Zevalin) targets ____1____ on B cells in non Hodgkins lymphoma (follicular lymphoma).

Murine IgG1 90Y conjugate licensed in UK 2002.

Delivery of cytotoxic ____2____ leading to death of the cell.

Also induces ADCC and CDC.

A
  1. CD20
  2. Radiation
156
Q

Mabs to treat allergy

One of the most promising therapeutic approaches in allergy is the neutralization of IgE by antibodies directed against the region of IgE involved in ____1____ with IgE receptors.

Such antibodies inhibit IgE action by preventing IgE binding to FceR bearing cells and may inhibit new IgE production by targeting IgE switched ____2____ cells.

A
  1. Interaction
  2. B
157
Q

Non-anaphylactogenic IgE antibodies

The concept of an inhibitor that blocks binding of IgE to FceRI but lacks ability to trigger ____1____ was realised by generating a monoclonal antibody which binds IgE at the same site as FceRI.

By virtue of this binding specificity the mAb has the ability to ____2____ IgE effector functions by blocking IgE binding.

This antibody rhuMAb-E25 is a recombinant humanized (>95%) monoclonal antibody (IgG1) that binds to free IgE.

Mab also does not induce IgE cross linking and activation of IgE sensitized mast cells as the receptor masks the ____3____ on FceRI bound IgE.

A
  1. Degranulation
  2. Inhibit
  3. Epitope
158
Q

In vivo application of anti-IgE antibodies in animals can result in long term IgE unresponsiveness. This is presumably through clonal elimination of IgE switched B cells by ____1____. Only B cells switched to IgE are affected.

These antibodies are well tolerated in man:

Phase I trials - 96% reduction in free IgE and 96% downregulation of FceRI on basophils (220 000 receptors to 8 000 receptors)

Phase II trials - Significant ____2____ in nasal symptoms in allergic rhinitis and early and late phase response in allergic asthma.

A
  1. Apoptosis
  2. Reduction
159
Q

Read

Clinical Trials Omalizumab (Xolair)

Studies involving 4300 patients with severe persistent asthma who were inadequately controlled despite receiving standard therapy.

  • Xolair reduces the rate of exacerbations (worsening of symptoms) by 38% versus placebo
  • The total number of emergency visits was reduced by 47%.
  • Xolair reduces the need for inhaled corticosteroids
  • Xolair improves asthma symptoms, lung function and quality of life.
  • Xolair reduced the rate of asthma attacks by 55%
A
160
Q

Mabs designed for the specific treatment of infectious diseases are unusual but there is one currently on the market.

Palivizumab (synagis) is a chimeric monoclonal antibody against the fusion protein of respiratory syncitial virus (RSV). Palivizumab blocks ____1____ of RSV into the cells.

Licensed in 1998 is given monthly (for 5 months) to children especially those with risk of developing severe ____2____ due to RSV (prematurity (<35 weeks gestation), congenital cardiopathies, chronic respiratory disorders.

A
  1. Entry
  2. Pathology
161
Q

Read

The Future of Therapeutic Monoclonal Antibodies

It is unlikely that antibody therapy will ever be the single therapy available in the treatment of cancer. However it is likely that antibody therapy will be used in combination with either chemotherapy, radiotherapy or surgery to whittle the cancer cell population to zero.

Main growth areas are antibodies conjugated with radioisotopes and toxins although they may have unpredictable and little understood side effects. Also bispecific antibodies that are designed to two different antigens are another growth area.

Though treatment with MAbs is still very expensive, there is much work going on in the development of transgenic plants or genetically altered goats milk for the production of antibodies.

Although still many problems latest financial predications suggest that major companies such as Abbott, Schering Plough and Pharmacia will each see revenues in excess of £350 million from the sale of MAb.

A
162
Q

Bispecific antibodies can have many more ______ 1 ______than monomeric antibodies because they interfere with two separate systems.

Same with all multimeric.

But there is the chance to have a ____2____ therapeutic effect because of this.

A
  1. Side effects
  2. Greater
163
Q

In N-glycosylation the glycan is bound via a N-glycosidic bond to Asparagine within the consensus amino acid sequence (sequon).

Name the sequon sequence.

A

Asn-X-Ser/Thr

(where X can be any amino acid other than Pro or Asp).

Medical Biotechnology (7 decks)

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