11/5/20

Phage Display in Drug Discovery and

Development
Next…….

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Phage Display in Drug Discovery and

Development
Ø What might we use this technology/methodology for?

Ø Most drugs work by interacting with receptors, genes or pathways

Ø Proteins, for example, are chiral environments, composed of amino acids linked together in peptide
chains. This means they are perfectly suited to binding peptides

Ø The cost of producing large peptide libraries is low

Ø Often better biocompatibility

Ø Peptides therefore make good drugs

Ø Predicted that therapeutic peptides will reach 45,542 million USD by 20241

1) https://www.zionmarketresearch.com/report/ peptide-therapeutics-market

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Peptides as Drug Targets

Ø Peptides typically refer to a chain of amino acids of around 50 residues or less

Ø Very useful for studying protein-protein interactions due to their high selectivity for the targets

Ø Can interfere with or stimulate receptor-ligand binding; altering or restoring metabolic processes

Ø Peptides can be used for drug delivery and a range of other drug-related functions

A useful approach to drug discovery involves:

• Identifying a particular target (receptor, pathway or gene) that is involved in the disease development and
progression

• This is followed by HTS of combinatorial chemical libraries of small molecules, allowing the identification of a
selection of target-specific compounds (a reverse pharmacology approach)
target-based drug discovery. This
approach involves identifying a particular
Phage Display is the widely used in vitro approach totarget
reverse
(e.g., a pharmacology
receptor, pathway, or gene)
that is involved in disease development
and progression. This is followed by high-
throughput screening of chemical libraries
of small molecules, allowing for the iden-
tification of a pool of target-specific com-
pounds. The reverse pharmacology
78 approach has led to a shift towards a
combinatorial approach in which high-
throughput in vitro screening of different
types of peptidic or proteic libraries is
performed against a selected biological
target. Among the currently available in
vitro display technologies, phage display
technique, M13 bacteriophages are
genetically modified to expose (display),
on their surface, random small peptides
fused with the minor coat protein pIII (five
copies/phage) or major coat protein pVIII
(2800 copies/phage) [4]. It is then pos-
sible to obtain, on a large scale, a highly
diversified group of phage clones, each
one expressing a random sequence.
The power of phage display comes from
two distinctive features: (i) the establish-
ment of a physical connection between
the phenotype (the displayed peptide)
and the genotype (the DNA sequence
encoding the displayed peptide) within
the same viral particle; and (ii) the pro-
the M13 filamentous phage, la
groundwork for the field [5]. F
Smith’s achievements, Gregory
exploited the phage display meth
to produce antibodies for the trea
diseases such as multiple scler
cancer [6]. The first antibody
with this methodology was ada
(HUMIRA), which was approved b
2002 for treatment of rheumatoid
psoriasis, and inflammatory bo
eases [7]. Smith and Winter e
shared a Nobel Prize for Che
2018 for their pioneering work
confirms the importance
technology.

Peptides as Drug Targets is undoubtedly the most widely used.

Overview of Phage Display

duction of large and diversified libraries
of peptides displayed on the surface of
phage particles.
Selection of Target-Specifi
Peptides
Let’s consider: Phage display is a powerful tool that The preparation of phage display l
allows researchers to identify and isolate George P. Smith was the first to success- the first step in the procedure o
peptides with high affinity and specificity fully display recombinant peptides at the binding selection known as bio
Ø A population of bacteriophages each with a distinctforphenotype in the form of a random peptide displayed on the
the target of interest. In this N-terminal end of the pIII capsid protein of The general principle of this p
surface of the phage (because it has been encoded on the coat protein gene)
Titering
Target-coated
Ø The bacteriophages are subjected to a selective Repeat micro!ter plate well
3–4 !mes
pressure by exposing them to the target molecule Incuba!on with phage
display library
and subsequent washing
(A)
(D)
Ø Only the phages that bound to the surface will
remain and will go on and reproduce, by being
allowed to reinfect a population of bacteria. This (E)
transmits their genotype to the next generation ELISA to determine
specificity of binding
a"er 3–4 rounds

(F)
(C)
(B)

Elute Wash out unbound

bound phages phages

Figure 1. Workflow of In Vitro Biopanning. The biopanning procedure involves six principal steps for phage selection and peptide characterization. (A) T
presented to the phage-displayed library allowing binding. (B) After incubation, unbound and nonspecific phages are washed out. (C) Bound phages are rec
an elution step. (D) The eluted phages are amplified by Escherichia coli reinfection and the previous steps are repeated three or four times. (E) At the end of

79 the phage titer is evaluated. (F) Eluted phage clones from the last round of panning are tested to determine their specificity of binding to the target by

88 Trends in Pharmacological Sciences, February 2019, Vol. 40, No. 2

2
division and established medicines business.
These changes will leave breast cancer drug
brance (palbociclib) and pneumococcal
vaccine Prevnar 13 doing most of the heavy
ifting. Future attempts to climb back up
he polls might be hindered by what many
ee as a lack of pipeline innovation from
he pharma giant, and recent management
promises of new gene therapies will take time
o get to market.
There has also been movement among
he ranks of last year’s biggest selling drugs
FIG. 1b), with Keytruda usurping Celgene’s
only gives it the mega-blockbuster Botox, but
also the scale to move up next year’s company
sales rankings.
Finally, while other companies held their
relative positions at the bottom half of the
table last year, Takeda was a new entrant 11/5/20
following its $62 billion takeover of Shire,
the largest acquisition of a foreign company
in Japanese history.
Lisa Urquhart
Vantage, London, UK.
e-mail: LisaU@vantageanalysis.com
https://doi.org/10.1038/d41573-020-00047-7

Top selling drugs 2019

Revlimid (lenalidomide) to claim the number
wo spot. Much of Keytruda’s impressive
Competing interests
The author declares no competing interests.

b Product (company)
48.2 Humira (AbbVie) 19.2
44.6 19.9
46.0 Keytruda 11.1
43.5 (Merck & Co.) 7.2
43.9 Revlimid (Celgene) 9.7
45.3 9.7
40.9 Eliquis 7.9
37.4 (Bristol-Myers Squibb) 6.4
40.0 Opdivo 7.2
38.9 (Bristol-Myers Squibb) 6.7
0 Avastin (Roche) 7.1 Humira
1 7.0
Rituxan (Roche) 6.5 Keytruda
6.9
Stelara 6.4
Opdivo
(Johnson & Johnson) 5.2 Avastin
6.1
2019
Herceptin (Roche) 7.1 2019 Rituxan
2018 Prevnar 13 (Pfizer) 5.8 2018 Stelara
5.8
Herceptin
35 40 45 50 0 2 4 6 8 10 12 14 16 18 20
Sales (US$ billions)

9. a | Top ten companies by sales of prescription and over-the-counter drugs. *Data for Takeda are
ly. Data for 2018 are included for comparison. Source: EvaluatePharma.

www.nature.com/nrd
Nature, 228 | April 2020 | volume 19

80

Antibodies as Drug Targets

https://www.cytivalifesciences.com/en/us/solutions/bioprocessing/knowledge-center/optimizing-purification-of-antibody-fragments
Protein Targeting Compounds, DOI 10.1007/978-3-319-22473-2_3

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Antibodies as Drug Targets

Fabs are considered the first genera.on of an.body fragments. They were originally generated by cleavage of an intact
an.body using an enzyme, such as papain, but are now produced using modern gene.c engineering approaches. Papain
cleavage yields two monovalent Fab fragments, each composed of one variable heavy chain (VH) and one variable light
chain (VL), linked by disulfide bonds and displaying a single an.gen-binding site.
scFvs are monovalent structures, with affinity for a single an.gen. With an approximate size of Mr 25 000, an scFv
contains the variable regions of an an.body’s heavy and light chains fused into a single polypep.de chain via a short
flexible linker. An scFv comprises the complete an.gen-binding site of its parental an.body molecule.
dAbs are some of the smallest func.onal an.body fragments that retain full an.gen-binding specificity, as they consist of
the VH or VL domains. The dAb is approximately one-tenth of the molecular weight of a normal an.body. Although dAbs
contain only three of the six complementary determining regions from the parent an.body, they exhibit an.gen binding
specificity and affinity. A dAb can be remarkably stable under harsh condi.ons of temperature, pressure, and denaturing
chemicals.

Protein Targeting Compounds, DOI 10.1007/978-3-319-22473-2_3

82

Antibodies as Drug Targets

Protein Targeting Compounds, DOI 10.1007/978-3-319-22473-2_3

83