Download Microbial Production of Therapeutic Agents:

 Microbial Production of Therapeutic Agents:
 Before the development of recombinant technology, most
human protein pharmaceuticals were available in:
 Limited quantities
 They were costly to produce and
 In a number of cases, their biological mode of action was not
well characterized
 After the development of recombinant DNA technology, it
was heralded/indicated as a means of producing a whole range
of possible human therapeutic agents in sufficient quantities for
both efficacy testing and eventual human use
 This forecast has turned out to be true
 Today, the genes mostly cDNAs for over 1,000 different
proteins that are potential human therapeutic agents have been
cloned
 Most of these sequences have been expressed in host cells and
currently around 500 are undergoing clinical trials for the
treatment of various human diseases
 More than 250 of these “biotechnological drugs” have been
approved for use in the United States or the European Union
Table 10.1 Glick 4th Ed
 However, it will be several years before many of the other
proteins are commercially available, because medical products
must first be tested rigorously in animals and then undergo
through human trials before being approved for general use
 It has been estimated that in 2006 the annual global market for
human recombinant protein drugs was about $ 60 billion and
ten “block buster” drugs constitute nearly half of these sales

In 2006, rituzimab (Rituzan), a monoclonal antibody
used to treat individuals with non-Hodgkin lymphoma,
generated nearly $ 4 billion in sales

While various forms of recombinant insulin generated
around $ 2.5 billion
 The development of
 treatments
 preventive procedures and
 cure for human diseases was the outstanding contribution
of medicine and science to human well being in 20th century
 This process, however, is a continuous one
 So called old diseases like tuberculosis can reappear if
preventive measures are relaxed or if resistant organism arise
 The idea of using antibodies as therapeutic agents has come to
fruition in past several years and specific antibodies are being
tested to attack:
 toxins
 bacteria
 viruses or even
 cancer cells
 An antibody may be viewed as a target-seeking missile or as a
magic bullet that either can directly neutralize an offending
agent or
 if equipped with a warhead or poison arrow can destroy a
specific target cell
 Unfortunately, despite their theoretical promise, the use of
antibodies for preventing or treating diseases and other
conditions has been limited
 However, recently, the development of recombinant DNA
technology and means of producing monoclonal antibodies has
combined with rapidly growing understanding of the molecular
structure and functions of immuno-globulin molecules to
reactivate interest in using specific antibodies to treat various
diseases
 Pharmaceuticals:
 Engineering Human Interferon (IFN);
 Interferons can be classified into three different groups on
the basis of chemical and biological properties
•
IFNα and IFNβ:
i.
Synthesized in cells that have been exposed to
viruses/viral DNA
ii.
•
IFNα encoded by a family of 15 non-allelic
genes while IFNβ by a single gene
IFN-γ: Synthesized in response to cell growth
stimulating agents and encoded by a single gene
 IFNα, β and γ have subtypes with different specificities For
Example:
• Anti viral activities of IFNα2 and α1 approximately
same when assess with a virus challenged bovine cell
line but
•
IFNα2 is seven times more effective than IFNα1 when
human cells are treated with virus
•
IFNα2 30 times less effective than IFNα1 when mouse
cells are used in this assay
Table 10.3 Glick 2nd Ed
Attempts have been made to engineer IFNs with combined
properties
In one study, hybrid genes from IFNα2 and α3 were
constructed in an effort to create proteins with novel IFN
activities
Fig. 10.2 Glick 3rd Ed
Hybrids expressed in E. coli
Resultant proteins were purified and examined for various
biological functions
Greater activity then parental molecules noted when tested
for the extent of protection of mammalian cells in culture
Many of hybrid IFNs induced test cells to synthesize 2’-5’
oligoisoadenylate synthetase
 This enzyme generates 2’- 5’ oligoisoadenylate which, in
turn, activates a latent cellular endoribonuclease cleave
viral mRNA
 Other hybrids had an anticancer activity against human
cancers
 This activity was greater then that of the parental molecules
 The creation of these hybrid interferon demonstrates that
new potential therapeutic molecules can be constructed by
combining functional domains from related gene
 Engineering Human Growth Hormones;
 The strategy of designing proteins by either functional
domain shuffling or site-directed mutagenesis can be used
to augment or constrain the mode of action of a protein
 Native human growth hormones (hGH) bind to both
growth hormones and prolactin receptors on a number of
different cell types
 To avoid unwanted side effects during therapy it is
desirable that hGH bind only to GH receptors
Fig. 10.3 Glick 3rd Ed
 Enzymes:
 DNase 1;
 Cystic fibrosis most common fatal hereditary disease
among Caucasians
 Approximately 30,000 diagnosed cases in USA and 23,000
in Canada and Europe
 It is estimated that a mutant cystic fibrosis gene is carried
by 1 in 29 Europeans, 1 in 65 African Americans and 1 in
150 Asians
 Individual with this disease are highly susceptible to
bacterial infection in their lungs
 Antibiotic treatment of patients who have these recurring
infections eventually leads to the selection of the antibiotic
resistant bacteria
 Presence of bacteria, some alive and some lysed causes the
accumulation of a thick mucus in the lungs of these patients,
making breathing very difficult
 A portion of the thick mucus in the lungs is the result of the
DNA that is released when bacterial cells are lysed
 To address this problem, Scientists at the USA
Biotechnology Company, Genentech isolated and expressed
the gene for the enzyme DNAse 1 which can hydrolyzed
long polymeric DNA chain into much shorter
oligonucleotides
 Then they delivered the purified enzyme in an aerosol to the
lungs of the patients with cystic fibrosis
 What DNAse treatment has done?
 The DNAse treatment acted to decrease the viscosity of
the mucus in the lungs and made it easier for these patients
to breathe
 While this treatment is not cure for cystic fibrosis, it
nevertheless relieves the most severe symptoms of the
disease in most of patients
 This enzyme has been approved in 1994 by USA Food and
Drug Administration
 Sale is predicted approximately $ 100 million per annum
by around the year 2000
Fig 17.3 Brum
Fig 17.7 Glick 3rd Ed
 Monoclonal Antibodies as Therapeutic Agents:
 Today with the advent of
hybridoma methodology,
antibodies are once again seen as potential therapeutic agents
 In fact, a number of monoclonal antibodies have been
approved for treating human diseases
Table 10.3 Glick 4th Ed
 This technique can be used to maintain a continuous supply
of pure mono-specific antibody
Fig 10.13 Glick 3rd Ed
complementaritydetermining regions
Papian - Two Fab and one Fc
 Preventing Rejection of Transplanted Organisms:
 In the 1970s, passive immunization was considered as a way of
preventing immunological rejection of a transplanted organ
 The rationale was to administer a specific antibody to the
patients that would bind to certain lymphocytes and diminish
the immune response directed against the transplanted organ
 In this effort, OKT3, the mouse monoclonal antibody was first
approved in 1986 by the USA Food and Drug Administration
 For use as an immunosuppressive agent after organ
transplantation in humans
 Mechanism:
 Lymphocytes that differentiate in the thymus are called T
cells
 Various members of the T-cells population act as immunological helper and effector cells are responsible for organ
rejection
 OKT3 binds to cell surface receptor CD3 which is present
on all T cells
 This action prevents a full immunological response and
spares transplant organ from rejection
 Immuno-supression by this means was reasonably effective
but there were some side effects including fever and rash
formation as anticipated. Why….?
 Chemically Linked Monoclonal Antibodies:
 Drugs that are very effective when tested in vitro – usually in
culture- are often much less potent in vivo
 This apparent loss of potency is due to the drug’s not reaching
its targeted site at a concentration sufficient to be effective
 Increasing the dose of a drug is not the answer to this problem,
because high drug concentrations often have deleterious side
effects
 Moreover, to avoid unwanted side effects, many drugs
administered at lower than optimal levels, thereby decreasing
their efficacy
 A number of different strategies may be used to enhance the
delivery of a drug to its target site:
 Drug may be encapsulated in liposomes i.e. particles in
which drug is surrounded by a specific lipid surface, that
can be targeted to certain organs
Fig 2.17 Lodish 3rd Ed
Fig 10.14a Glick 3rd
Fig 10.14b Glick 3rd
 The majority of natural death in North America and Europe are
the result of blockage of a cerebral/coronary artery by the blood
clot i.e. thrombus
 A thrombus consists of a network of fibrin, a blood clotting
agent, which is formed in response to defect in the walls of a
blood vessel
 Under natural conditions, plasmin degrades the fibrin in a
blood clot and dissolves the clot
 Plasmin is a serine protease and is produced by the activation
of plasminogen by plasminogen activator
 In many instances, however, arterial blockage occurs when
this biological system does not remove blood clots efficiently
 Thus, it was reasoned that plasminogen activators could be
used as therapeutic agents to induce higher levels of plasmin,
which in turn, would efficiently remove the arterial thrombi,
thereby reducing their impact on brain and heart arteries
Fig 10.15 Glick 3rd Ed
Fig 10.16 Glick 3rd Ed
 Human Monoclonal Antibodies:



Although the initial studies of immunotherapeutic agents were
promising, there are drawbacks to chemical coupling and the
use of a nonhuman monoclonal antibody
Not only are the yields of chemically linked molecules low but
the coupling occurred at random sites
The chemical coupling procedure may also inactivate the
enzyme activity of the plasminogen activator or other protein
that may be used
 Finally as noted previously, if the therapy requires multiple
treatments, the antibody component should be from human
source to prevent immunological cross-reactivity and
sensitization of the patient
 It is very difficult to create specific non-cross reactive antibodies because of problems associated with obtaining human
monoclonal antibodies by conventional hybridoma techniques
 Therefore, other approaches for obtaining human monoclonal
antibodies were devised:
 It may possible to introduce cells of the human immune system
into mutant mouse strain that lacks for most parts, its own
natural immunological cell repertoire
 Transplantation of human immune system stem cells, such a
mouse, with severe combined immunodeficiency (SCID
mouse)
 As a result mouse acquires a human cell immune system and
 In response to challenge by antigen, can produce human
antibodies

 It may also to introduce human immunoglobin genes into
the germ line of mice to create transgenic mice that would
produce a human immunoglobin after immunization with
particular antigen
Both methods are laborious. Therefore, genetic engineering is
now used to create:
 to create both human therapeutic antibodies and
 effective dual-function proteins that have the ability to
bind a target and then destroy it
 Hybrid Human – Mouse Monoclonal Antibodies:
 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 antigenbinding specificity
 This hybrid molecule is called a chimeric antibody or
humanized antibody depending which portion of the mouse
antibody are remove
Fig 10.17 Glick 3rd Ed
Fig 10.18 Glick 3rd Ed
Fig 10.19 Glick 3rd Ed
 To date, with this procedure, more than 50 different monoclonal
antibodies have been humanized
 While this technology is clearly effective and widely applicable
but time consuming and expensive
Single-Chain Antibody;
 In addition to producing Fv fragment, attempts have been made
to determine whether a single protein chain consisting of only VL
and VH domains would form a functional antigen binding
molecule?
 Computer simulations of 3-dimensional structure of a potential
single chain antibody showed that VL and VH domains have to
be separated by a linker peptide to assume the correct
conformation for antigen binding
Example:
- Humanized antibody directed against
the surface of human colon cancer cells
was tested in patients with colorectal
cancer
- Remained in blood system about 6X
longer
- Expended the period of effectiveness
- Only one patient/10 showed mild
response
- Starting with a rodent Hybridoma cell line, cDNA for the L and H can be isolated.
- The variable regions of the cDNAs can be amplified by PCR
 On the basis of these design constraints, DNA constructs 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
 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
 Single chain antibody may be used for a variety of therapeutic
and diagnostic applications in which Fc effector's functions are
not required and
 When a small size is an advantage as speeds antibody uptake
and facilitates a more even distribution in solid tumors
Fig 10.25a Glick 3rd Ed
Fig 10.25b Glick 3rd Ed
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