Transcript Chapter 10

Protein Therapeutics
Protein Therapeutics
 Prior to recombinant DNA, protein
pharmaceuticals were very difficult and
expensive to produce. They were also available
in very limited quantities and their biological
modes of action were not well understood.
 Several thousand different potentially therapeutic
proteins were cloned and expressed in both
mammalian and bacterial host cells.
 More than 250 of these “biotechnology drugs”
have been approved for use in the U.S. or E.U.
 Examples of “biotechnology drugs” have been
approved for human use in the U.S. or E.U.
 Antibodies can be used as theraputic agents to
target specific cell to be destroyed.
Pharmaceuticals
 Interferons: a class of proteins produced
largely by virus infected cells that are secreted
to the bloodstream and activate resistance
pathways in other cells.
Pharmaceuticals
 Potential use as a drug in many disorders (MS,
hepatitis, etc.)
 Produced in three forms in humans (IFN-α, IFNβ, and IFN-γ) with different biological activity.
 IFN-α and IFN-β, are produced as a result of
exposure to viruses, encoded by a family of 13
different genes and two genes, respectively.
 IFN-γ is as a result of growth stimulating
factors, encoded by a single gene.
 Overview of protocol used to isolate IFN cDNA.
 Isolated cDNAs are clones into expression
vectors for production in E. coli.
 Interferon gene shuffling.
 In human INFs, researchers found that the level
of anti-viral activity varied greatly within the
subtype.
 IFNα-2 and IFNα-3 had common restrictions sites
allowing the production of hybrid genes to create
proteins with novel IFN activities.
 Enhanced production of oligoisoadenylate
synthetase.
 It produces 2’-5’ linked oligonucleotides that
activate a latent endoribonuclease which attacks
and degrades viral mRNAs.
 Hybrid genes construction.
 Others had antiproliferative activities against
human cancers.
 Longer-acting interferons.
 Fusing an IFN gene with the gene for a stable
protein, human serum albumin, produces a
stable hybrid protein.
 Human growth hormone: stimulates tissue
and bone growth, increases protein synthesis
and mineral retention, & decreases body fat
storage.
 Effective in treatment of dwarfism, infants and
children who lack sufficient endogenous
levels of human growth hormone, chronic
renal insufficiency, and Turner’s syndrome.
 Human growth hormone was one of the first
therapeutic proteins approved for human use.
 The first recombinant growth hormone was
called somatrem.
 The modification of the native protein was
done to eliminate side effects.
 It is desirable that growth hormone will bind
only to growth hormone receptors but not
prolactin receptors.
 Site-directed mutagenesis was used to alter 3
amino acids which bind zinc ions (His-18, His21, and Glu-174).
 These modifications yielded human growth
hormone derivatives that bound only to
growth hormone receptors.
 These derivatives are being tested for safety
and efficacy in humans.
 It is advantageous to have a long-lasting form.
 The receptor fragment is fused to growth
hormone resulting in dimerization which in
turn stabilizing the growth hormone in vivo.
 Another method to produce a long-lasting
human growth hormone - Albutropin.
 The N-terminous of human growth hormone is
fused to the C-terminous of human serum
albumin .
 Tumor necrosis factor alpha (TNF-α): a potent
antitumor agent, but not used widely due to
severe toxicity.
 If the protein could be delivered to the site
(tumor), it could be used in lower doses and the
unwanted side effects would be diminished .
 Adding 6 amino acids targeting peptide to the Nterminus results in TNF-α with tumor specificity.
 In mice, this fusion was 12-15X more effective,
and much higher percentage of survival.
 This work is preliminary until it can be tested in
humans.
 A 6-amino-acid targeting peptide (red) fused
to its N terminous.
 Survival of lymphoma-bearing mice following
treatment with native and modified TNF.
Therapeutic enzymes
 Dnase I: used in the treatment of cystic fibrosis
patients to relieve the severe symptoms by
decreasing the viscosity and adhesivity of
mucus in the lungs, made it easier to breathe.
Therapeutic enzymes
 Actin binds very tightly to Dnase I and inhibits its
ability to cleave DNA.
 Changing one amino acid (either ala to arg or tyr
to arg) decreases actin binding and increases
activity 10-50X.
 Alginate lyase: also used in cystic fibrosis.
 Cystic fibrosis patients often have
Pseudomonas aeruginosa infected in the
lungs which is impossible to treat with
antibiotic treatment due to the biofilm.
 It secretes an alginate-like polysaccharide that
increases viscosity of mucus.
 Alginate lyase can liquefy bacterial alginate,
together with or prior to antibiotic treatment,
significantly decreased the number of bacteria
in biofilm.
 This result suggests that, in addition to the
Dnase I treatment, alginate lyase would help.
 Isolated alginate lyase gene from
Flavobacterium and cloned into E. coli for
screening.
 cDNA was cut to process a lower MW enzyme
with higher specificity that easily hydrolyzes
bacterial alginates.
 DNA construct encoding the 40,000-Da alginate
lyase is fused to the leader peptide from a B.
subtilis α-amylase gene.
 The construct is under the transcriptional
control of a B. subtilis penicillinase gene
expression system.
 When these transformants were grown in liquid
medium, the recombinant alginate lyase was
secreted into culture broth.
 Phenylalanine ammonia lyase (PAL)
 Phenylketonuria (PKU) results from a defect in
phenylalanine hydroxylase.
 This enzyme produces tyrosine from
phenylalanine. Results in mental retardation
due to a build up of phenylalanine.
 Treatment entails a controlled semisynthetic
diet with low levels of phenylalanine through
infancy and possibly for life.
 The administration of the enzyme is an
alternative treatment but it is not very stable
and requires a cofactor for activity.
 Treatment with PAL degrades phenylalanine
to ammonia and trans-cinnamic acid.
 The experiment in mice indicated the lower
levels of plasma phenylalanine.
 α1-Antitrypsin: Many pathogens use human
proteases in processing their proteins.
 A therapeutic agent that blocked its activity
might act as a broad-spectrum antiviral and
antibacterial agent.
 When tested in culture, a variant of alphaantitrypsin blocks the production of HIV type 1
glycoprotein gp160, measles virus protein F0,
and human cytomegalovirus.
 Clinical efficacy is as yet unknown.
 Could be very useful due to variety of viruses
inhibited.
 The antitrypsin will
block the proteolytic
activity preventing
the production of
infectious pathogen.
 Glycosidases: Possible use in preparing blood
for transfusions.
 ABO blood types correspond to specific
carbohydrates on surface of red blood cell.
 A = N-acetylgalactosamine
 B = galactose
 O = neither
 There are specific glycosidases that cleave
these sugars from the surface and convert type
A, B, and AB into type O.
 Digestion of monosaccharides that determine
blood groups A and B to obtain blood group O.
 Lactic acid bacteria are widely used in the
production and preservation of fermented foods.
 Lactic acid bacteria-many used as a probiotic
that claim to offer a health benefit by altering the
indigenous microflora of the intestinal tract.
 They can be used to treat several gastrointestinal
disorders, including lactose intolerance.
 Lactobacillus lactis has been developed as a
host system for cloning and expressing proteins
to be delivered to the human gut.
 The treatment of ulcerative colitis and Crohn’s
diseases is to lower the levels of cytokines, TNF-α.
 Interleukin-10 secreting L. lactis show promise in
alleviating bowel inflammation in victims of Crohn’s
disease and ulcerative colitis.
 To prevent any plasmid-borne antibiotic
resistance marker genes spreading to other
bacteria in the environment, the construct is
inserted into L. lactis chromosomal DNA by
homologous recombination.
 The recombinant bacteria grew well in the
laboratory and produce interleukin-10 when
thymine or thymidine was added.
 Leptin secreting L. lactis are a possibility for
treating severe obesity. It has been shown to
reduce food intake and body weight loss in
obese leptin-deficient mice.
 Leptin was produced in L. lactis without
formation of inclusion body and was secreted.
 Nisin is a 34-amino-acid-residue polycyclic
peptide that has antibacterial activity.
 HIV inhibitor: Cyanovirin N (isolated from Nostoc
cyanobacteria) is an inhibitor of HIV infection.
 It is possible that Cyanovirin N secreting Lactobacillus
jensenii could be used as a topical microbicide to help
prevent HIV infections in women.
Monoclonal antibodies as
therapeutic agents
 Antibodies were first used to treat disease
about 100 years ago in diphtheria with a
polyclonal horse serum raised against
Cornyebacterium diphtheriae.
 It generally was effective if used in the first few
days of infection, but often sensitized
individuals for a second treatment causing
anaphylactic shock and death because patients
often develop antibodies against the foreign
proteins.
 It is now possible to engineer antibodies with a
greatly reduced level of immunogenicity in humans.
 A continuous supply of pure monospecific antibody
can be maintained.
 Problems with cross-reactivity and anaphylaxis
though have still recurred to a lesser extent.
 Human monoclonal antibodies with both specific
immunotherapeutic properties and lowered
potential for immunogenicity have been produced.
 See Table 10.3 and Boxes 10.2 & 10.3.
 Structure & function of antibodies: Each
antibody is made up of 2 identical light and
heavy chains held together by both hydrogen
and precisely localized disulfide linkages.
 The N-terminal portion of the L and H chains
together determine antigen recognition site.
 The site consist of three complementarity
determining regions (CDR’s) that lie within the
variable regions at the N-terminous.
 The Fab fragment retains the antigen-binding
activity after digested with the proteolytic
enzyme papain.
 In fact, the N-terminal half of the Fab, the Fv,
contains all the activity.
 The Fab portions can be used in treatments since
they maintain the same binding but do not elicit
some of the same responses.
 The Fc portion elicits several immunological
responses after antigen-antibody binding.
 The compliment cascade-breaks down
membranes, activates phagocytes, and signals
to the rest of the immune response.
 Antibody-dependent cell-mediated cytotoxicity
(ADCC)-as a result of binding Fc portion to
receptors on ADCC effector cells. These
release compounds that lyse foreign cells.
 Fc portion binds to receptors on phagocytes
causing them to engulf the antibody-antigen
complex.
 Preventing rejection of transplanted organs:
 The idea was to use a specific antibody that
bind to certain lymphocytes diminishing
immune response against the transplanted
organ.
 Mouse monoclonal antibody OKT3 was
approved as an immunosuppressive agent.
 OKT3 binds the CD3 receptor on T-cells and a
full immunological response is blocked.
 Mouse monoclonal antibodies show some
problems including poor coupling to
drugs/proteins as well as sensitization.
 Due to difficulties producing human
monoclonal antibodies, hybrid mouse/human
monoclonal antibodies have been produced.
 These are produced by genetically
engineering mouse myeloma cells to produce
modified human antibodies.
 The chimeric antibody with the antigen-
binding specificity of the mouse monoclonal
antibody diminishing immunogenicity and
introducing human Fc effector capabilities.
 The humanized antibody replacing the CDRs
from the mouse in human antibody. The
product has antigen-binding specificity of the
mouse and all other properties of human
antibody.
 The primers complementary to the rodent
CDR with 5’ ends that complementary to
human antibody. Then, by PCR, the amplified
rodent CDRs are spliced into human antibody.
 Generation of a
Xenomouse that
can synthesize
only human Ig.
They are selected,
immunized, and
used to make
hybridomas
producing human
antibodies.
 IgG is the main antibody found in mammalian
serum, and it is native form that is used in
therapeutic antibodies.
 A variety of IgG derivatives or fragments that
may be used instead of whole antibody molecule.
 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 toxin or
some other specific activity to a cell.
 Most antibody-toxin combinations have been
constructed using the gene for Pseudomonas
endotoxin A.
 It is 66-kDa protein with 3 domains; binding,
translocation, and ADP-ribosylation.
 An immunotoxin is made by replacing the Nterminal binding domain from the gene with
the single-chain antibody sequence.
 The molecules are very similar in size to the
original toxin with the ability to bind, enter,
and kill specific cell.
 These hybrids bind specific cells and allow
the toxin to penetrate easily.
 Toxin molecules may also be directed to
cancer cells by using a dispecific diabody that
is engineered to bind to a surface-specific
tumor-associated antigen and then to a toxin.
 It is possible to create peptides that are smaller
than scFvs and still retain the ability to bind to a
specific antigen.
 They are more likely to penetrate a tumor and
stop the tumor growth.
 Next, the peptide is coupled to a toxin molecule.
 Production of antibodies
in E. coli that acts as
“bioreactors.”
 Done by cloning H&L
genes, combining them in
phage vectors, screening,
excising the exons as part
of a plasmid, and
transform E. coli.
 Harvest antigen-binding Fv
fragment from E. coli.
 DNA constructs of an Fv combinatorial gene
library cloned into bacteriophage λ DNA,
thereby creating all possible combinations of L
and H chain fragments.
 Production of
antibodies in
the filamentous
bacteriophage
M13.
 A combinatorial library of antibody fragments
displayed on the surface can be screened by
ALISA-like system.
 This approach is easier than using plaque assay.
 A combinatorial library of full-length antibodies
were expressed in periplasm of E. coli with the
antibody anchored to the inner membrane.
 Screening is done by fluorescently labeled
antigen.
 Chemically-linked monoclonal antibodies:
Many potentially useful drugs in vitro fail in
vivo. This is often due to the inability to
deliver the drug to the target cell in a
sufficient concentration.
 Increasing the dose of drug is not the answer,
because high drug concentrations often have
deleterious side effects.
 Strategies have been developed to overcome
this problem using monoclonal antibodies
conjugated to the drug or to an enzyme which
activates the drug.
 A monoclonal
antibody-based drug
delivery system.
 Dual-variable-domain immunoglobulin containing
two tandem Fv regions, each with a different
specificity.
 Anticancer antibodies may also bind to some non-
cancerous cell.
 This approach presents researchers with only a
limited number of targets for therapeutic
antibodies.
 Tumor cell are first treated with the
chemotherapeutic agent irinotican, which induces
the synthesis of a unique cell surface protein.
 Then, a monoclonal antibody directed against the
cell surface protein and conjugated to a toxin
molecule is added.
 The toxin is internalized after the binding, thereby
killing the tumor cell.