Chapter 5

Chapter 5
Protein Therapeutics
UNIVERSITY OF BALAMAND
FACULTY OF SCIENCES
DEPARTMENT OF BIOLOGY
BIOL 287
BIOTECHNOLOGY AND RECOMBINANT DNA
Takla El Khoury, Ph.D.

Molecular and Cell Biology
Biology Department
Faculty of Sciences
Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

of Recombinant DNA, Fifth Edition American Society for Microbiology
Bernard R. Glick, Cheryl L. Patten 1752 N St. NW, Washington, DC 20036-2904
Chapter 5
➢Most human protein pharmaceuticals were available in only limited quantities, they
were extremely costly to produce, and, in a number of cases, their biological modes of
action were not well characterized.
➢When recombinant DNA technology was first developed, it was used as a means of
producing a whole range of human therapeutic agents in sufficient quantities for both
efficacy testing and eventual human use.
➢This estimation has turned out to be true. Today, the genes for several thousand
different proteins that are potential human therapeutic agents have been cloned.
➢Most of these sequences have been expressed in mammalian as well as bacterial host
cells, and currently nearly 1,000 are undergoing clinical testing with human subjects
for the treatment of various diseases.
➢ Approximately 300 of these "biotechnology drugs” have been approved for use in
the United States or the European Union

Chapter 5
Table 5.1

Chapter 5
-Pharmaceuticals
➢Protein therapeutic agents may be divided into several different categories based on
the general mode of action of the therapeutic agent
➢ In this regard, a large number of the currently available protein therapeutic agents that
are produced by recombinant DNA technology replace a deficient or abnormal protein
➢ Other recombinant therapeutics may enhance an existing pathway or provide a novel

function or activity.
➢Some classic examples of replacing a deficient protein include providing diabetics

with insulin or hemophiliacs with specific blood coagulation factors.

Chapter 5
-Human Interferons
➢Interferons (IFNs) are animal glycoproteins that are synthesized by cells in response to
various pathogens, especially viruses.
➢IFNs "interfere" with viral replication within host cells. IFNs can also :
oactivate immune cells, such as natural killer cells and macrophages;
oincrease recognition of infected or tumor cells by upregulating antigen presentation to T

lymphocytes
oIncrease the ability of uninfected host cells to resist new infection by an infected virus
➢IFNs may induce the production of hundreds of other proteins, upregulate major
histocompatibility molecules, activate cells that are part of the immune system, promote
apoptosis, and inhibit protein synthesis of both viral and host genes.
➢At the present time, 12 different IFNs and IFN derivatives have been approved by the
FDA for the treatment of various diseases
Chapter 5 -Synthesis In E. coli of a Polypeptide with Human
Protein Therapeutics Leukocyte Interferon Activity
➢The genes for several IFNs have been Isolated, and clinical trials have shown that they
are effective treatments for a variety of viral diseases.
➢Several research groups have engineered IFNs with combined properties based on
different members of the IFN- gene family that vary in the extents and specificities of
their antiviral activities.
➢This can be achieved by splicing a portion of one IFN-  gene with a DNA sequence
from a different IFN- gene to create, after translation, a hybrid protein that exhibits
novel properties (properties different from either of the contributing genes).
➢In one study, hybrid genes from IFN- 2 and IFN- 3 were constructed in an effort to
create proteins with novel IFN activities.
➢When tested for the extent of protection of mammalian cells in culture against viral
infection, some of the hybrid IFNs were found to have greater activity than the parental
molecules.
Chapter 5
Protein Therapeutics -Clinical trials
➢lnfact, some hybrid IFNs have recently undergone successful clinical trials and
have been approved for use as human therapeutic agents.
➢The strategies for creating hybrid IFNs can also be applied to other human gene
families whose products have therapeutic potential
➢Clinical trials are conducted In three distinct phases, generally requiring a total of
about 7 to 9 years at a cost of approximately $100 million to $400 million to
complete.
➢At each stage, various compounds are dropped from consideration based on the
results obtained.
➢This slow and expensive process Is claimed to be the most effective method ever
devised to assess the efficacy of a treatment (Zlvin, 2000).

Chapter 5
Protein Therapeutics -Clinical trials
➢The three phases of the FDA review process areas are:
Phase I: With between 10 and 100 healthy people, the safety of the drug and, starting
with very low doses, the highest dosages that can be administered are assessed. To
check if there is a chance that serious side effects may result.
Phase II: With 50 to 500 affected patients, the optimal dosing schedule (regimen) is
determined. A control group is used so that it is possible to dearly distinguish between
the effects of the drug and the natural remission of the disease. The use of a control
group also helps to mark out real from apparent side effects of the treatment.
Phase III: Depending upon the disease, approximately 300 to 30,000 patients who
have the disease are tested. After It is established that the drug is not harmful (phaseI)
and the optimal dosing regimen has been determined (phase II), the effectiveness of the
treatment needs to be proven.

Chapter 5
Protein Therapeutics -Human Interferons
➢Therapeutic agents that maximize early antiviral response

Figure 5.2 and maintain viral suppression throughout the course of
therapy have the best chance of achieving lasting
eradication of the virus from an infected individual
➢One approach to creating long-acting IFNs includes

pegylation of the therapeutic agent
➢The process of pegylation entails covalently attaching the

chemical polyethylene glycol to proteins (PEG).
➢This binding is typically achieved by Polyethylene glycol

incubation of a reactive derivative of polyethylene glycol
with the target protein molecule.
➢As a consequence of pegylation, the effective size of

modified IFNs in solution increases, thereby prolonging the
circulatory time of the therapeutic protein by reducing its
renal (kidney) clearance.
Chapter 5
Protein Therapeutics -Human Growth Hormones
➢Human growth hormone (somatotropin) is a 191-arnino-acid pituitary protein that

stimulates the production of insulin-like growth factor 1.
➢Infants and children who lack sufficient endogenous levels of human growth hormone
respond to treatment with growth hormone, which stimulates tissue and bone growth,
increase protein synthesis and mineral retention, and decreases body fat storage
➢Human growth hormone was one of the first recombinant therapeutic proteins in the
world to be approved for human use
➢Treatment of children entails daily subcutaneous injections during the years when the
child is growing.
➢The cost of the treatment varies depending on the country and the size of the child but
varies between $40,000 to $50,000 per year.
➢In addition to children with growth hormone deficiency, in 2004, FDA approved the use
of recombinant human growth hormone for individuals whose short stature was caused by
a variety of medical conditions other than human growth hormone deficiency.
Chapter 5
Figure 5.3
➢Since the current treatment regimen is expensive,
it would be advantageous to have a long-lasting
form of human growth hormone.
➢DNA encoding the extracellular domain of the

human growth hormone receptor was fused to
human growth hormone cDNA using a DNA
fragment encoding 20-amino-acid-long linker
peptide consisting of four repeats of the amino acids
Gly4Ser
➢This construct has a very strong tendency to

dimerize as the growth hormone moiety from one
molecule binds with the receptor portion of another
molecule.

Chapter 5
Figure 5.3
➢It is thought that the dimerization of the growth
hormone construct stabilizes human growth
hormone in vivo so that it is cleared from plasma
approximately 300 times more slowly than free
monomeric human growth hormone.
➢Under these conditions, the active monomeric

form (A) is slowly released from the inactive
dimeric growth hormone (B), allowing it to bind
to the growth hormone receptor (C).
➢More recently, when a fusion protein that did

not include the peptide linker was constructed and
tested, this protein yielded growth hormone levels
that were 1.7 times higher than when the linker
was a part of the construct.

Chapter 5
Figure 5.3
➢Therefore, all subsequent studies including

human trials have involved the fusion protein
without a peptide linker.
➢Since the fusion protein is a single peptide that

does not require any additional manipulation, the
costs of its preparation compared to pegylated
growth hormone are significantly reduced.
➢This unique modified release fusion growth

hormone protein is currently in phase II clinical

Chapter 5
➢When this growth hormone construct was tested in rats, a single injection promoted
growth for five days (120 hours) compared to the usual requirement in rats for (at
least) daily injections.
Figure 5.4

Chapter 5
Protein Therapeutics -Tumor Necrosis Factor Alpha
Figure 5.5 ➢Tumor necrosis factor (TNF) is a cytokine (a cell signaling

molecule) whose primary role is regulation of the immune
system.
➢TNF is produced as a 212-amino-acid-long transmembrane

protein arranged instable homotrimers that are subsequently
cleaved by the metalloprotease TNF-converting enzyme to
form a soluble homotrimeric cytokine TNF.
➢While a number of studies have clearly shown that tumor

necrosis factor alpha (TNF-) is a potent antitumor agent, it
has not been widely used in this capacity because of its severe
toxicity.
➢If TNF-  could be delivered directly to its intended site of

action (the tumor), then lower doses could be used and the
unwanted side effects would be reduced.

Chapter 5
Protein Therapeutics -Tumor Necrosis Factor Alpha
➢To develop a version of TNF- with tumor

Figure 5.5
specificity, DNA encoding the peptide CysAsn-
Gly;Arg-Cys-Gly(i.e.,cystein-asparagine-glycine-
Survival of mice with lymphoma
arginine-cysteineglycine) which targets a particular
tumor cell surface protein, was fused to TNF-
DNA
➢The fusion protein contained a 6-amino-acid

extension at its N-terminal end.
➢In mice, the cytotoxic activities of Cys-Asn-Gly-

Arg-CysGly-TNF- and TNF- were identical,
indicating that the additional amino acids did not
reduce toxicity.
➢However, the modified version of TNF- was 12

to 15 times more effective at inhibiting tumor
growth than the unmodified form.

Chapter 5
Protein Therapeutics -Extending Protein Half-Life
➢The most common strategies for extending the in vivo half-life of therapeutic proteins
are pegylation and the fusion of the therapeutic protein with human serum albumin.
➢ However, each of these approaches has limitations. For example, while pegylation
may both reduce the immunogenicity and increase the stability of therapeutic proteins,
it may significantly increase the costs of the therapeutic protein, and in some cases
pegylated proteins or their metabolites may accumulate in the kidney therefore
interfering with normal kidney functioning
➢ Similarly, fusion proteins that include human serum albumin do not always behave as
expected.
➢Thus, scientists have sought to determine whether an unstructured synthetic protein that
is covalently attached to the target therapeutic protein might provide some of the benefits
of pegylation and fusion to human serum albumin without any of disadvantages

Chapter 5
Protein Therapeutics -Extending Protein Half-Life
➢In designing such a protein, it was decided to exclude:

oall hydrophobic amino acids (which of ten lead to compact structures or protein
aggregation).
oAmide-containing aminoacids that may become unstable upon long-term storage
o positively charged side chains that may cause binding to membranes
o cysteine residues that become either oxidized or cross-linked

➢Ultimately, only six amino acids were used in the design of the unstructured protein (i.e.
alanine, glutamic acid, glycine, proline, serine, and threonine).
➢A DNA library encoding randomized 36-amino-acid peptides was constructed and

screened for expression level.
➢Highly expressed sequences were ligated and then rescreened for maximal activity

Chapter 5
Protein Therapeutics -Enzymes
-DNase I
➢Cystic fibrosis is one of the most common

Figure 5.7
fatal hereditary diseases.
➢Individual with cystic fibrosis are highly

susceptible to bacterial infections in their
lungs.
➢Antibiotic treatment of patients who have

these recurring infections eventually leads to
the selection of antibiotic-resistant bacteria.
➢The presence of bacteria, some alive and

some lysed, contributes to the accumulation
of a thick mucus in the lungs of these
patients, making breathing very difficult and
acting as a source for further infection.

Chapter 5
Protein Therapeutics -DNase I
Figure 5.7
➢The thick mucus in the lungs is the result of
the combination of the alginate that is
secreted by the living bacteria, the DNA that
is released from lysed bacterial cells, and
degenerating leukocytes that accumulate in
response to the infection, as well as
filamentous actin derived from the
cytoskeletons of damaged epithelial cells
➢To address this problem, scientists at the

U.S.biotechnology company Genentech
isolated the gene for the human enzyme
deoxyribonucleaseI (DNaseI) and expressed
the gene in CHO cells in culture.
➢DNase I can hydrolyze long polymeric

DNA chains into much shorter
oligonucleotides.
Chapter 5
➢The purified enzyme was delivered in an

Figure 5.7
aerosol spray to the lungs of patients with
cystic fibrosis.
➢The DNase I decreased the viscosity and

adhesivity of the mucus in the lungs and
made it easier for these patients to breathe.
➢While this treatment is not a cure for cystic

fibrosis, it nevertheless relieves the most
severe symptom of the disease in most
patients.
➢The monomeric form of actin binds very

tightly to DNase I which significantly
inhibits its ability to cleave DNA. This
interaction limits the effectiveness of DNase I
as a therapeutic agent.

Chapter 5
➢Scientists al Genentech predicted which
Figure 5.8 amino acid residues of DNAse I interacted with
actin and were therefore possible targets for
changing by directed mutagenesis.
➢For example, changing amino acid 144 from

alanine to arginine or amino acid 65 from
tyrosine to arginine decreased the binding of
DNase I to actin up to 10,000-fold.
➢In addition, the actin-resistant mutants had 10-

to 50-fold more DNase I activity than the native
enzyme.
➢In addition to its use with cystic fibrosis

patients, the modified DNase I may be used to
treat chronic bronchitis, pneumonia,
bronchiectasis, emphysema, asthma,

Chapter 5
Protein Therapeutics -Alginate Lyase
➢Alginate is a polysaccharide polymer that is produced by a wide range of seaweeds

and both soil and marine bacteria.
➢It is composed of chains of the sugars -D-mannuronate and -L-guluronate. The

two residues on the left are -(1-4)-linked L-guluronic acid, while the two residues on
the right are -(1-4)-linked D-mannuronic acid
➢The cross-linked alginate polymer forms an elastic gel.
Figure 5.9

Chapter 5
➢The excretion of alginate by mucoid strains of the bacterium Pseudomonas

aeruginosa that infect the lungs of patients with cystic fibrosis significantly
contributes to the viscosity of the mucus in the airways.
➢Once mucoid strains of P. aeruginosa have become established in the lung of cystic
fibrosis patients, it is almost impossible to eliminate them by antibiotic treatment.
➢This is because the bacteria form biofilms in which the alginate prevents the
antibiotics from coming into contact with the bacterial cells.
Figure 5.10

Chapter 5
➢In one experiment, it was shown that the

Figure 5.10 addition of the enzyme alginate lyase, which
can liquefy bacterial alginate, together with or
prior to antibiotic treatment, significantly
decreased the number of bacteria found in
biofilms.
➢Thus, alginate lyase treatment not only

decreases the viscosity of the mucus but also
facilitates the ability of added antibiotics to kill
the infecting bacterial cells.
➢This result suggests that in addition to the

DNase I treatment, decomposition of the
alginate might help clear blocked airways of
individuals with cystic fibrosis.

Chapter 5
Figure 5.11
➢An alginate lyase gene has been isolated from a

Flavobacterium species, a Gram-negative soil bacterium
that is a strong producer of this enzyme.
➢A Flavobacterium genomic DNA library was constructed

in E. coli and screened for alginate lyase-producing clones
by plating the entire library onto lid medium containing
alginate.
➢Following growth, colonies that produced alginate lyase

formed a halo around the colony when a solution of calcium
was added to the plate

Chapter 5
➢Analysis of a cloned DNA fragment from one of
Figure 5.11 the positive colonies revealed an open reading
frame encoding a polypeptide with a molecular
mass of approximately 69,000 Da.
➢Detailed biochemical and genetic studies

indicated that this polypeptide is a precursor of the
three different alginate lyases produced by the
Flavobacterium sp
➢After the 69,000-Da precursor is produced, a

proteolytic enzyme cleaves off an N-terminal
peptide of about 6,000 Da.
➢The resultant 63,000-Da protein can lyse both

bacterial and seaweed alginates.
Cleavage of the 63,000-Da protein yields a 23,000-Da enzyme that depolymerizes

seaweed alginate and a 40,000-Da enzyme that is effective against bacterial alginate.

Chapter 5
➢To produce large amounts of the 40000-Da enzyme, the DNA corresponding to the
enzyme was amplified by the PCR and then inserted into a Bacillus subtilis plasmid vector
fused to a B.subtilis -amylase leader peptide to direct the secretion of the protein and
placed under the transcriptional control of a penicillinase gene promoter
➢Further tests showed that the enzyme produced using this construct efficiently liquefied
alginates that were produced by mucoid strains of P. aeruginosa that had been isolated
from the lungs of patients with cystic fibrosis.
Figure 5.12

Chapter 5
➢Unfortunately, since most sources of alginate lyase are
Figure 5.13 nonhuman, these proteins are not necessarily a good
choice for use as a therapeutic agent, as they likely to
generate an immune response in the patient, thereby
causing a range of complications.
➢To significantly decrease the antigenicity of the

alginate lyase, the enzyme may be chemically modified
with polyethylene glycol (PEG)
➢However, nonspecific modification of alginate lyase

with PEG results in a large decrease in the activity of the
enzyme.
➢Nevertheless, it is possible to avoid the inactivation of

the enzyme while still decreasing its antigencity by
specifically modifying alginate lyase with PEG To do
this several specific mutants of alginate lyase were
created by directed mutagenesis in which one amino
acid residue at a time was changed to cysteine
Chapter 5
Protein Therapeutics -Phenylalanine Ammonia Lyase
Figure 5.14
➢The human genetic disease phenylketonuria is an
autosomal recessive trait resulting in the impaired
functioning of the enzyme phenylalanine hydroxylase
➢A possible treatment for this disease would be the

administration of the enzyme phenylalnine hydroxylase
which should be pegylated to reduce its immunogenicity.
➢When phenylalanine hydroxylase, which oxidizes

phenylalanine to tyrosine, is deficient, the normal
development of an individual is impaired and mental
retardation results due to a buildup of phenylalanine
➢0n the other hand, phenylalanine ammonia lyase,

which converts phenylalanine to ammonia and trans-
cinnamic acid is a stable enzyme that does not require a
cofactor and could potentially prevent the accumulation
of phenylalanine in phenylketonuria patients.
Chapter 5
Protein Therapeutics -Phenylalanine Ammonia Lyase
Figure 5.14
➢ The cinnamic acid is largely converted to hippuric
acid and then excreted in the urine
➢Experiments in mice showed that phenylalanine

ammonia lyase is an effective substitute for
phenylalanine hydroxylase, and the orally delivered
enzyme is sufficiently stable to survive the mouse
gastrointestinal tract and still function.
➢However, with repeated administration of nonhuman

phenylalanine ammonia lyase, this enzyme is
immunogenic and its continued use becomes
problematic.
➢Thus, this enzyme was pegylated to reduce its

immunogenicity suggesting that it may have potential
as a novel therapeutic agent for the treatment of
phenylketonuria.
Chapter 5
Protein Therapeutics -Targeting Mitochondria
➢ The human mitochondrial genome consists of circular DNA approximately 16 kb
encoding 37 separate proteins.
➢ Moreover, more than 1,000 additional proteins that function within the mitochondrion
are encoded within the nuclear DNA synthesized in the cytosol and then imported into
the mitochondria across the characteristic double membrane.
➢ The nucleus-encoded proteins are synthesized with a mitochondrial targeting

sequence, which allows them to be imported into the mitochondria.
➢ Once a protein is inside, the mitochondrial targeting sequence is recognized and

cleaved off, allowing the protein to function without any disorder.
Figure 5.20

Chapter 5
➢ Most mitochondrial disorders (encoded within the nuclear DNA) appear as neurological
disorders, but they can also manifest as myopathy (muscle disease), diabetes, multiple
endocrinopathy (endocrine disease and hormone imbalance)
➢ Enzyme replacement therapy might also be an effective strategy for mitochondrial

disorders provided that a simple and effective means of directing enzymes to the
mitochondrion can be found.
➢ One possibility is to synthesize the target enzyme as a fusion protein with the 11-amino-
acid-long arginine- and lysine-rich transcriptional activator of transcription (TAT)
peptide (facilitates passage through cell membrane)
Figure 5.20

Chapter 5
➢ TAT fusion proteins may be rapidly and efficiently introduced into cultured cells, intact
tissues, and live tissues (when injected into mice) while retaining the biological activity
of the target protein
➢ The lipoamide dehydrogenase (LAD), subunit of three multicomponent enzyme

complexes in the mitochondrial matrix including pyruvate dehydrogenase, is responsible
for the metabolism of carbohydrates and amino acids. A defect in LAD results in
extensive metabolic disturbances in an affected individual.
➢ The ability of the TAT peptide to help to deliver human LAD enzyme into human cells
in culture was examined. LAD enzyme was efficiently delivered into cells in culture and
to their mitochondria, restoring LAD activity to normal values
Figure 5.20
MTS: mitochondrial transit sequence

Chapter 5
Protein Therapeutics -Lactic Acid Bacteria
➢ A probiotic is a live microorganism that is claimed to confer a health benefit by

altering the original microflora of the intestinal tract.
➢ Lactic acid bacteria have also been used to treat several gastrointestinal
disorders, including lactose intolerance, traveler's diarrhea, antibiotic-associated
diarrhea, infections caused by various bacterial and viral pathogens, and
immunopathological disorders, such as Crohn disease and ulcerative colitis.
➢ Not only are lactic acid bacteria easy to deliver orally, they also have the
potential to elicit both mucosal and systemic immune responses.
➢ In the past few years, lactic acid bacteria have been used as a host system to
express various foreign genes, with the idea that these bacteria facilitate the
delivery of the proteins encoded by the genes to the human gut

Chapter 5
Table 5.3

Chapter 5
Table 5.3 (Continued)

Chapter 5
Protein Therapeutics -Interleukin-10
➢ Ulcerative colitis and Crohn disease are both diseases of the intestinal tract
➢ Ulcerative colitis is associated with excess type 2 T helper cell cytokines,

including interleukin-4 and interleukin-5, whereas in Crohn disease, type 1T
helper cell cytokines, including TNF-α, IFN-α, and interleukin-2, are
overproduced.
➢ The treatment for Crohn disease often includes trying to lower the levels of
cytokines, especially TNF-α.
➢ One approach has been the administration of antibodies against TNF-α. Other
workers have targeted interleukin-10 as a means of controlling Crohn disease
because it modulates the regulatory T cells that control inflammatory
responses to intestinal antigens.
➢ However, interleukin-10 is not clinically acceptable because it needs to be

administered by frequent injections.
➢ To overcome this problem, the bacterium L. lactis was engineered to

synthesize and secrete interleukin-10.
Chapter 5
➢ Experiments were performed with mice
Figure 5.21 to test whether interleukin-10 secreting
L. lactis could be used to treat
inflammatory bowel disease.
➢ First, interleukin-10-secreting L. lactis

was fed to mice with ulcerative colitis
that had been induced by the addition of
5% dextran sulfate to their drink water.
➢ Second, strains of mice that are

genetically incapable of synthesizing
interleukin-10 and provide an animal
model for ulcerative colitis were tested
➢ In both of these cases, the engineered L.

lactis significantly alleviated the
symptoms of the disease, establishing
that this approach works in principle
Chapter 5
Figure 5.21
➢ One concern about the use of an interleukin-10 secreting L. lactis strain as a therapeutic
approach is the possibility that the genetically modified bacterium will be released to the
environment.
➢ If this were to happen, the plasmid carrying the interleukin-10 gene and any plasmid -
borne antibiotic resistance marker genes could be spread to other bacteria in the environ
ment.
➢ To prevent this from occurring, a synthetic human interleukin-10 gene that replaced the
L. lactis thymidylate synthase gene, thyA, which is essential for the growth of the
bacterium, was inserted into the bacterial chromosome of L. lactis by homologous
recombination
Chapter 5
Figure 5.21
➢ This strain produced interleukin-10 and grew well in the laboratory when either
thymidine or thymine was added to the medium.
➢ However, when it was deprived of thymidine and thymine, the viability of the bacterium
declined by several orders of magnitude.
➢ When this modified bacterium was tested in pigs, whose digestive tract is similar to that
of humans, it propagated well and actively produced interleukin-10.
➢ In addition, laboratory experiments demonstrated that the modified L. lactis was

extremely unlikely to acquire a thymidylate synthase gene from other bacteria in the
environment, confirming both the safety and efficacy of this approach
➢ Recently, phase II clinical trials with this interleukin-10-secreting L.lactis strain were
initiated
Chapter 5
Protein Therapeutics -Leptin
➢ It has been estimated that approximately 30% of the North American and 20% of the
European populations are overweight
➢ However, it is possible that real weight reduction may be obtained by administration

of the protein leptin.
➢ Leptin, the product of the obese (ob) gene, is a 167-amino-acid protein with a
molecular mass of approximately 16kDa.
➢ Leptin is synthesized as a precursor with a 21-amino-acid-long signal peptide that is

removed when leptin is secreted.
➢ Treatment with recombinant leptin can reduce food intake and correct metabolic
perturbations in (homozygous) leptin-defident mice.

Chapter 5
➢However, when it is introduced subcutaneously leptin is not particularly effective in

obese patients unless their serum leptin concentrations reach levels 20- to 30-fold higher
than normal.
➢This poor response has been attributed to the inefficient transport of leptin across the
blood-brain barrier.
➢To overcome this problem, intranasal delivery of leptin has been devised
➢The nasal mucosa is highly vascularized, so delivery of a thin layer of medication

across its broad surface area can result in rapid absorption of the medication into the
bloodstream

Chapter 5
➢When leptin is produced in E. coli, it typically

Figure 5.22 forms insoluble inclusion bodies that must be
solubilized and renatured before the active
protein is generated.
➢Therefore, the 462-bp cDNA for human leptin

was cloned with its signal peptide and expressed
under the control of the nisin promoter in L. lactis
➢In L. lactis, leptin was produced efficiently

without the formation of an inclusion body and was
secreted from the recombinant bacteria.
➢When the leptin-producing L. lactis strain was

administered intranasally in obese mice, the mice
significantly reduced their food intake and body
weight. This approach opens up the possibility that,
if delivered properly, leptin might act as an
effective weight loss treatment in humans.
Chapter 5
Protein Therapeutics -An HIV Inhibitor
➢The compound cyanovirin N, isolated from a cyanobacterium, blocks several steps of

HIV infection, preventing virus entry into human cells.
➢Cyanovirin N is a cyanobacterial lectin, a protein that binds strongly to specific

carbohydrate residues. This small two-domain protein neutralizes HIV by specifically
binding to high-mannose glycans (polysaccharides) on the viral surface envelope
glycoproteins well as to high mannose oligosaccharides found on the HIV envelope.
➢The binding of cyanovirin N to these carbohydrate residues thereby prevents

interaction of the virus with a mammalian host cell. Moreover, in addition to its activity
against HIV, cyanovirin N is also active against other influenza, Ebola, hepatitis C, and
some strains of herpesvirus.
➢Cyanovirin N can be expressed at a sufficiently high level in a vaginal strain of

lactobacilli

Chapter 5
Protein Therapeutics -Recombinant antibodies
➢Unfortunately, this kind of antibody therapy carries considerable risk and is not
widely used today. Patients often develop antibodies against the foreign proteins of
either whole or partially purified antiserum (from horse for example)
➢The use of antibodies as therapeutic agents is generally considered too dangerous

for patients and is used only rarely
➢However, with the development of hybridoma technology, antibodies are once again
being viewed as potential therapeutic agents. One reason for the renewed interest in
therapeutic antibodies is that it is now possible to engineer antibodies with a greatly
reduced level of immunogenicity in humans
➢ In addition this technique can be used to maintain a continuous supply of pure

monospecific antibody. However, the problems of cross-reactivity leading to an
immune response have not been completely overcome.
➢Thus, the recipient might still produce antibodies to a monoclonal antibody that
carries mouse (murine) determinants.

Chapter 5
➢To avoid this problem; human monoclonal antibodies with both specific
immunotherapeutic properties and lowered potential for immunogenicity have been
produced.
➢In fact, a number of monoclonal antibodies have been approved for treating human
diseases
Table 5.4

Chapter 5
Table 5.4 (Continued)

Chapter 5
Protein Therapeutics -Hybrid Human- Mouse monoclonal antibodies
Figure 5.26
➢The modular nature of antibody functions has made it
possible to convert a mouse monoclonal antibody into
one that has some human segments but still retains its
original antigen-binding specificity.
➢This hybrid molecule is called a chimeric antibody (A)

or, with more human sequences a "humanized" antibody
(B).
➢The difference between a chimeric and a humanized

mouse monoclonal antibody is the portion of the mouse
antibody that has been removed.
➢ In a chimeric antibody, the portion of the mouse

monoclonal antibody that was targeted for replacement
with a human sequence was the mouse Fc fragment
because it is the most likely fragment to cause the
production of human antibodies.
Chapter 5
Figure 5.26
➢To diminish immunogenicity and to introduce
human Fc effector capabilities, the DNA coding
sequences for the Fv regions of both the L and the H
chains of a human immunoglobulin were substituted for
the Fv DNA sequences for the L and H chains from a
specific mouse monoclonal antibody (A)
➢Chimeric antibodies are composed of approximately

70% human and 30% mouse DNA sequences
➢The humanizing of mouse monoclonal antibodies has

been taken one step further than the formation of
chimeric molecules by substituting into human antibodies
only the complementarity-determining regions
(CDRs) of the mouse monoclonal antibodies (B).
➢Humanized antibodies consist of approximately 95%

human and 5% mouse DNA sequences

Chapter 5
Figure 5.27
➢Humanized antibodies have antigen-binding affinities similar to those of the original

mouse monoclonal antibodies, they are more effective therapeutic agents and are less
likely to generate an immune response.
➢The humanizing of mouse monoclonal antibodies may be performed as follows

(Fig.).Starting with a mouse hybridoma cell line, cDNAs for the L and H chains (CDRs)
are isolated. The variable regions of these cDNAs are amplified by PCR.
➢The humanized variable-region cDNAs are then cloned into expression vectors, which
are then introduced into appropriate host cells, usually Chinese hamster ovary (CHO)
cells, for the production of antibodies.
Chapter 5
Figure 5.28
➢on 1 November 2013,the FDA approved the use
obinutuzumab for the treatment of CD20-positive B-cell non-
Hodgkin’s lymphoma.
➢Obinutuzumab is a fully humanized monoclonal antibody

that, like rituximab, is directed against the CD20 protein found
on B cells. Obinutuzumab is seen by Genentech, the
manufacturer of rituximab,as the commercial successor to
rituximab (a chimeric monoconal antibody).
➢Obinutuzumab is thought to target and destroy CD20-positive

B-cell by either directly binding to the tumor cell surface-
exposed CD20 molecules or through antibody dependent
cytotoxicity (ADCC) where the antibody recruits the immune
system to attack the CD20-positive B-cells (Fig).

Chapter 5
Figure 5.28
➢When obinutuzumab was compared directly with rituximab,

patients treated with obinutuzumab had significantly longer
periods of remission when they were disease free and did not
require treatment.
➢In addition, obinutuzumab has been glycol-engineered (its

complement of surface carbohydrates has been modified) so
that it does not contain any fucosylated sugar molecules.
➢It is thought that the lack of fucose moieties may result in an

increase in the ADCC activity of the antibody.

Chapter 5
Protein Therapeutics -Antibody Fragments
Figure 5.30 ➢Immunoglobulin (IgG) is the main antibody found in

mammalian serum, and it is the native form that is
almost exclusively used in therapeutic antibodies
➢The fact that IgG molecules have two identical sites

that bind to two identical antigens (they are bivalent)
generally increases their effectiveness In vivo.
➢While the Fc portion of the IgG molecule is

important in amplifying cascade of proteins that
➢Single-chain antibodies have a facilitates pathogen destruction, Fc-mediated effects
molecular mass of approximately 27 are not necessary for all applications and may even
kDa, compared with approximately sometimes be undesirable.
150 kDa for IgG molecules.
➢By manipulating portions of the IgG L and H-chain
➢Because of their small size, single- cDNAs, researchers have constructed a variety of IgG
chain antibodies can penetrate and derivatives or fragments that may be used instead of
distribute in large tumors more whole antibody molecule
readily than intact antibodies
Chapter 5
Figure 5.31
➢In addition, a protein-coding sequence can be linked to a single-chain antibody

sequence to create a dual-function molecule that can both bind to a specific target and
deliver a toxin or some other specific activity to a cell
➢DNA constructs of VL and VH sequences from a cDNA of a cloned monoclonal

antibody were each ligated to a chemically synthesized DNA linker fragment in the
order VL-linker-VH· (Fig A)
➢After expression in E.coli, the single-chain protein was purified, and both its affinity
and specificity were found to be equivalent to those of the original intact monoclonal
antibody.
Chapter 5
Figure 5.31
➢Moreover, instead of linking the VH and VL chains with a short peptide, amino acids
in the framework region (FR) can be modified to form a disulfide linkage between the
two peptides (Fig B).
➢The effectivenes of this disulfide-stabilized Fv molecule (VL-S-S-VH) coupled to a

cancer cell toxin was compared with that of an scFv molecule coupled to the same
toxin. The disulfide-stabilized and scFv immunotoxins had the same activity and
specificity.
➢However, the disulfide-stabilized immunotoxin was several fold more stable than the
scFv molecule. This result suggests that disulfide-stabilized Fv molecules may be
more useful than scFv molecules in some therapeutic applications
Chapter 5
Figure 5.32
➢One example of coupling a toxin to an antibody fragment is the fusion of a portion

of Pseudomonas exotoxin A to an scFv.
➢This toxin is a 66-kDa protein with three separate domains:
•Domain I is responsible for cell binding

•Domain II for translocation of the protein into the cell
•Domain III for ADP-ribosylation where the latter activity is responsible for the
toxicity of the molecule (Fig.A).

Chapter 5
Figure 5.32
➢An immunotoxin is generally synthesized by replacing the N-terminal domain of

the toxin (e.g., Pseudopionas exotoxin A (domain I)),with a single-chain antibody
sequence, thereby creating a molecule very similar in size to the original toxin with
the ability to bind, enter, and kill a specific cell (Fig.B).
➢A number of immunotoxins that have antitumor activity in vitro and in animal

models have been constructed.

Chapter 5
Figure 5.33
➢It is also possible to direct toxin molecules to cancer

cells by using a bispecific diabody that is engineered to
bind to a surface-specific tumor-associated antigen
and then to a toxin molecule, thereby directing the toxin
molecule to the tumor (Fig).
➢Several different engineered immunotoxins are

currently in clinical trials.

Chapter 5
Protein Therapeutics -Dual -Variable-Domain Antibodies
Figure 5.39
➢ These constructs are essentially IgG molecules containing two

tandem Fv regions, each with a different specificity.
➢ Dual variable-domain immunoglobulins are bispecific and

tetravalent and they consist entirely of human
immunoglobulin sequences.
➢ Each molecule contains four Fv regions, two identical Fv

regions directed against one antigen and two Fv regions
directed against another antigen.
➢ For example, a dual-variable-domain immunoglobulin specific

for both interleukin-12 and interleukin-18 produced in CHO
cells showed binding to both of these cytokines, binding each
with an affinity similar to that of the original monospecific
antibody
Chapter 5
Protein Therapeutics -Anticancer Antibodies
Figure 5.40 ➢ many therapeutic antibodies that are

directed against proteins that are
overexpressed on the surfaces of cancer
cells compared to noncancerous cells.
➢ For example, women with a type of breast

cancer that overproduces the human
epidermal growth factor receptor 2 (HER2)
protein on the surface of the tumor may be
effectively treated with the humanized anti-
HER2 monoclonal antibody trastuzumab
(Herceptin)
➢ One way to select for additional cell

surface targets would be to identify proteins
whose expression is selectively induced in
tumor cells exposed to chemotherapeutic
drugs.
Chapter 5
Figure 5.40 ➢ For example, when colorectal cancer cells were

treated with the drug irinotecan, which is a
topoisomerase enzyme inhibitor and is
commonly used to treat this of cancer, several
newly synthesized proteins were found on the
surfaces of those cells.
➢ The new cell surface proteins were expressed

early, after exposure to Irinotecan prior to any
major effects on the viability of treated cells.
➢ Monoclonal antibodies directed against one

newly synthesized cell surface protein (called
LY60/E48) were generated, and then the
antibodies were complexed with the cellular
toxin auristatin E. The antibody-toxin
conjugate was then used to treat tumor cells that
were first treated with irinotecan

Chapter 5
Figure 5.41 ➢ Some cancerous tumors can get around the immune-
checkpoint pathways by switching off the attack against
the tumor cells (B)
➢ This is particularly important for T cells that are specific

for tumor antigens. However, because many of the
immune checkpoints are initiated by ligand receptor
interactions, the tumor can be prevented from ruining the
functioning of the T cells by the addition of specific
antibodies directed toward the checkpoint receptor on
the T cell
➢ To be effective these antibodies must bind to the T-cell

checkpoint receptor and thereby prevent the tumor cells
from binding to the checkpoint receptor; in this case, the
T cells continue to be able to attack the tumor. This
approach is still in a relatively early stage of
development

Chapter 5
Figure 5.42
➢ To more effectively treat ovarian cancer it would be important to inhibit the proliferation
of ovarian cancer stem cells.
➢ To do this, researchers have first identified a number of proteins that are found uniquely
on the surface of these ovarian cancer stem cells.
➢ They have then systematically inhibited the expression of one or more of these proteins
in an effort to identify proteins that might be therapeutic targets for efforts to impair
ovarian cancer stem cell proliferation
Chapter 5
Figure 5.42
➢ One such protein is a tyrosine kinase-like orphan receptor 1(Fig.). Monoclonal

antibodies that are directed against this ovarian cancer stem cell surface protein inhibit
the cells ability to migrate and to seed new tumors, suggesting that these monoclonal
antibodies might provide an effective means of treating ovarian cancer patients.
➢ However, it remains to be demonstrated that this approach is as effective in patients

with ovarian cancer as it is in immune-deficient mice.

Chapter 5
Protein Therapeutics -Antiobesity Antibodies
KLH: Oxygen carrying metalloprotein
Figure 5.48
➢ The active form of the 28-amino-acid peptide ghrelin, which stimulates food intake, is
acylated on the hydroxyl side chain of serine 3 with n octanoic acid (Fig.A).
➢ In humans, ghrelin levels increase during dieting, with the unfortunate consequence that
ghrelin facilitates weight regain and therefore blocks sustained weight loss.
➢ It is believed that lowering the level of this peptide could reduce food intake and
promote permanent weight loss.
➢ To develop a catalytic antibody that is directed against ghrelin, chemically synthesized

transition state analogues of ghrelin are chemically coupled to keyhole limpet
hemocyanin (KLH) and then injected into mice
Chapter 5
Protein Therapeutics -Antiobesity Antibodies
Figure 5.48
➢ The inoculated mice then became the source of monoclonal antibodies that bind and
then specifically remove the n-octanoic acid moiety from ghrelin
➢ The ghrelin peptide in which the n-octanoic acid moiety is removed no longer has
biological activity and can no longer promote weight regain.
➢ Although this work is very preliminary, it is predicted that this catalytic antibody may
effectively lower ghrelin levels in humans and become an important helper to other
weight-loss strategies.

Chapter 5
The End !!


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BIOTECHNOLOGY AND RECOMBINANT DNA

Takla El Khoury, Ph.D.

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢ Other recombinant therapeutics may enhance an existing pathway or provide a novel

➢Some classic examples of replacing a deficient protein include providing diabetics

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

oactivate immune cells, such as natural killer cells and macrophages;

oincrease recognition of infected or tumor cells by upregulating antigen presentation to T

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢The three phases of the FDA review process areas are:

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢Therapeutic agents that maximize early antiviral response

➢One approach to creating long-acting IFNs includes

➢The process of pegylation entails covalently attaching the

➢This binding is typically achieved by Polyethylene glycol

➢As a consequence of pegylation, the effective size of

➢Human growth hormone (somatotropin) is a 191-arnino-acid pituitary protein that

➢DNA encoding the extracellular domain of the

➢This construct has a very strong tendency to

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢Under these conditions, the active monomeric

➢More recently, when a fusion protein that did

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢Therefore, all subsequent studies including

➢Since the fusion protein is a single peptide that

➢This unique modified release fusion growth

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

Figure 5.5 ➢Tumor necrosis factor (TNF) is a cytokine (a cell signaling

➢TNF is produced as a 212-amino-acid-long transmembrane

➢While a number of studies have clearly shown that tumor

➢If TNF-  could be delivered directly to its intended site of

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢To develop a version of TNF- with tumor

➢The fusion protein contained a 6-amino-acid

➢In mice, the cytotoxic activities of Cys-Asn-Gly-

➢However, the modified version of TNF- was 12

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢In designing such a protein, it was decided to exclude:

o cysteine residues that become either oxidized or cross-linked

➢A DNA library encoding randomized 36-amino-acid peptides was constructed and

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢Cystic fibrosis is one of the most common

➢Individual with cystic fibrosis are highly

➢Antibiotic treatment of patients who have

➢The presence of bacteria, some alive and

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢To address this problem, scientists at the

➢DNase I can hydrolyze long polymeric

➢The purified enzyme was delivered in an

➢The DNase I decreased the viscosity and

➢While this treatment is not a cure for cystic

➢The monomeric form of actin binds very

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢For example, changing amino acid 144 from

➢In addition, the actin-resistant mutants had 10-

➢In addition to its use with cystic fibrosis

Molecular Biotechnology: Principles and Applications Copyright © 2017 ASM Press

➢Alginate is a polysaccharide polymer that is produced by a wide range of seaweeds