Download Biotech applications – hybridomas and monoclonal antibodies

Introduction to immunology.
LESSON 6: BIOTECH AND BIOMEDICAL APPLICATIONS
Today we will get to know:
• A little bit of immunopharmacology and
immunotherapy
• Some examples of biotech applications of
immune system processes
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Introduction to immunology. Lesson 6
Immunopharmacology – how to suppress or stimulate the immune system
Immunopharmacology is a very wide term which we will use here to indicate those drugs that
act primary on the immune system. There are two major groups of immunopharmacological
drugs: immunosuppressive and immunostimulant drugs.
Immunosuppressive drugs
Immunostimulant drugs
Those drugs that suppress the immune system.
Particularly important for transplantation,
autoimmune disorders, allergies, and all the cases
where immune system is too active.
Those drugs that stimulate the immune system.
Particularly important for the treatment of
infectious diseases, tumors, immunodeficiencies and
all the cases where the immune system needs a
boost.
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Introduction to immunology. Lesson 6
Immunopharmacology – how to suppress the immune system
Immunosuppression is widely used in clinical practice. But it has some major problems: it
works better on primary responses than on secondary (memory) responses, it works best if
administered before exposure to the antigen, and shutting down the immune system is
always a risk.
Glucocorticoids
NSAIDs
Immunosuppressive
drugs
Antiallergic drugs
T-cell blockers
Cytotoxic drugs
Cell-based
Immunosuppression
(experimental)
Treg
tDCs
Antibody reagents
Anti-cytokine treatments
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Introduction to immunology. Lesson 6
Immunosuppressive drugs – the glucocorticoids
Glucocorticoids have a very wide range of effects on the immune system, and are thus widely
used as immunosuppressive drugs whenever significant inhibition of the immune system is
needed. Natural glucocorticoids are not used because of their significant mineralocorticoid
activity.
Glucocorticoids
like
prednisone,
prednisolone, methylprednisolone etc.
suppress both innate and adaptive
immune responses, at both the
humoral and the cellular level. But, to
do so, they need high doses.
The bad side is that their suppressive
activity lasts long (increased infection
risk), they can induce monocytopenia,
lymphopenia and eosinopenia, and
they have strong systemic side-effects
(glucose intolerance, bone dissolution,
muscle waste, Cushing’s syndrome)
Nature Rev. 2012
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Introduction to immunology. Lesson 6
Immunosuppressive drugs – the NSAIDs
To overcome side-effects of corticosteroids, Non-Steroidal Antinflammatory Drugs (NSAIDs) have
been developed. Regardless of their differences, they all inhibit ciclooxygenase (COX)-1 and -2
enzymes, which metabolize arachidonic acid to produce prostaglandins. NSAIDs are not
prototypical immunosuppressive, as they target many other cell types.
NSAIDs inhibit COX-2, which, under the
influence of inflammatory cytokines,
produces
inflammatory
prostaglandins. These, in turn, recruit
phagocytes to the inflamed sites,
stimulate fever and sensitize neurons to
pain.
The bad side is that they also inhibit
COX-1, which is ubiquitous and has
protective roles in the mucosae.
Suppression of COX-1 is associated to
high liver toxicity, gastric ulcerations
and higher frequency of gastric
carcinomas.
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Introduction to immunology. Lesson 6
Immunostimulation and immunotherapy
Immunotherapy deals with the idea of boosting an individuals’ immune system, to allow it
to destroy microbes and tumors. The boost can be biological (microbial-derived products),
pharmacological, or cell-based.
Immunostimulant
drugs
Microbial products
and
cytokines
STRONGER IMMUNE SYSTEM
STRONGER DEFENSES
Adoptive cell
transfer
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Introduction to immunology. Lesson 6
Immunostimulant drugs – microbial products
Many bacterial products are PAMPs, and they strongly stimulate inflammation by triggering
cytokine production in APCs. These, in turn, stimulate the adaptive immunity and, overall,
increase leukocytes number by boosting hematopoiesis.
Brandy Urology,
J.H. Hospital
The Bacillus Calmette–Guérin (BCG) is an
attenuated (less virulent, but still alive)
mycobacterium bovis strain. This is able to infect
human cells, but not to induce any pathology.
Rather, it can stimulate the production of Igs by
B-cell and thus behave as a vaccine against
mycobacterium tuberculosis. It has a strong
inflammatory effect on some tissues, and has
thus been also approved as a treatment for
bladder cancer.
The bad side is that PAMPs can induce massive
cytokine production, which can result in fever
and shock. This is especially true with cytokines,
which can also have direct toxic effects.
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Introduction to immunology. Lesson 6
Immunotherapy – cytokine therapies
Since cytokines control the whole immune system, and mostly stimulate it, it is logical to use
them whenever there is the need to boost immune system activity. They are used in clinical
practice, but they are burdened by severe side effects.
Cytokine therapies
Cytokine
Effects
Used for
Side effects
IL-2
Proliferation of T
cells,
differentiation to
Th and CTLs,
empowers innate
immunity
Melanoma,
metastatic renal
cancer
Toxic for blood
vessels, blood
leakage, edema,
hemorrhage, mental
disorders
IFN-a
Strong stimulation
of innate and
adaptive
immunity,
activation of the
antiviral defense
state
CGD, Kaposi’s
sarcoma, hairy cell
leukemia,
papillomavirus
infections,
hepatitis C,
multiple sclerosis
Flu-like syndromes at
every injection, pain,
diarrhea, anorexia,
confusion,
depression, suicide
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Introduction to immunology. Lesson 6
Immunotherapy – active vaccination
Active vaccination is the process of injecting individuals with microbial antigens, heat-killed
microbes or attenuated living microbes to induce antibody production and memory B-cells
formation. Thus, the individual acquires the ability to respond to the microbe he/she has
been vaccinated against.
To ensure memory B-cells formation, whole
microbes (either killed or attenuated) are
preferred, as they also trigger fever and
inflammation that boost B-cells activation and
memory cells formation.
Attenuated microbes are those living strains
which are still able to infect an individual, but
that generate a less-dangerous pathological
manifestation,
which
is
generally
inflammation/flu.
Side-effects are generally low, but sometimes
they can be extremely severe.
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Introduction to immunology. Lesson 6
Immunotherapy – adjuvants for vaccination
Injecting individuals with microbial antigens can determine unsufficient responses because
natural antigens are often weak. Also, injecting a living microbe can be dangerous, and
reducing the amount of microbe to the minimum is mandatory. Here’s the need for
adjuvants.
Types of adjuvants
Inorganic compounds
Alum, aluminum hydroxide,
aluminum phosphate, calcium
phosphate hydroxide
Mineral oil
Paraffin oil
Bacterial products
Killed bacteria like B. Pertussis, M.
Bovis
Organic compounds
Squalene
Detergents
QuilA
Cytokines
IL-2, IL-1, IL-12, TNF-a
Combined
Freund’s adjuvant (mineral oil + M.
Bovis)
Adjuvants are chemical or biological
products that can either boost Tcells, activate inflammation or help
to stabilize the antigen so that it can
stimulate B-cells for a longer time.
Adjuvants have been also implicated
in
clinical
side-effects
of
vaccinations, like the onset of
juvenile diabetes in the case of
Freund’s adjuvant.
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Introduction to immunology. Lesson 6
Immunotherapy – passive vaccination
In passive vaccination, individuals are injected with preformed immunoglobulins (from
donors). Thus, individuals acquire pools of immunoglobulins (good for immunodeficiencies)
and the ability to respond to certain microbes.
Transfusion of immunoglobulins is called
intravenous immunoglobulins (IGV). Generally,
IGVs contain IgG and IgA.
Anti-microbial IGVs are used against hepatitis
B, botulism, diphtheria, tetanus, rabies.
It’s generally well-tolerated, but sporadic sideeffects can be extremely severe.
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Introduction to immunology. Lesson 6
Immunotherapy – adoptive cell transfer
Adoptive cell transfer deals with the idea of isolating immune cells from individuals, expand
them in culture and then re-infuse them. This is a wonderful strategy to kill tumors.
Tumor-infiltrated lymphocytes (TIL) are a
heterogeneous population which includes
Th and CTLs able to recognize and kill the
tumor. The problem is that the cytokines
released from the tumor suppress them.
Taking these “good cells” out of the tumor
mass and re-injecting them upon expansion
strongly increases the ability of the immune
system to react against tumors.
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Introduction to immunology. Lesson 6
Immunotherapy – cell-based vaccination
Also dendritic cells can be “prepared” in vitro to show tumor antigens, and then re-injected
into the patient to stimulate tumor antigens’ recognition and tumor killing. This is currently
defined as cell-based vaccination.
5) The DC matures and is
infused back to the patient
3) The antigen is given to
DC
2) The antigen is purified in the
lab
4) The antigen is
uptaken by DC
6) The DC displays tumor
antigen and activates the
T-cells
1) Blood is drawn
7) T-cells attack cancer cells
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Introduction to immunology. Lesson 6
Biotech applications – hybridomas and monoclonal antibodies
Often, antigens are polyclonal (contain different epitopes), thus being able to stimulate the
expansion of different B-clones, each producing specific antibodies against a part of the
antigenic molecule. Overall, the mixture of antibodies produced is polyclonal.
Single epitopes
Single B-clones
Y
Antigen
Epitope
2
Epitope
3
Y
Y
Epitope
1
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Introduction to immunology. Lesson 6
Biotech applications – hybridomas and monoclonal antibodies
But tumors of B-cells (myeloma or plasmacytoma), originating from a single B-clone, can only
produce antibodies against a single epitope. Hence, they are monoclonal.
Y Y
Y
Y
Neoplastic
transformation
Y
B-cell tumor
Single B-clones
Y
Single epitopes
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Introduction to immunology. Lesson 6
Biotech applications – hybridomas and monoclonal antibodies
This suggested that it was theoretically possible to generate monoclonal B-cells, immortalize
them (so they can be cultured in the labs like tumor cells) and use them to produce large
amounts of the desired antibody.
In 1975, G. Kohler and C. Milstein described the method to generate
antibodies with a single specificity (monoclonal), by fusing normal Bcells from immunized lab animals with myeloma cells. The chimera
formed, being a cell able to produce antibodies and to survive in culture
like a tumor, was called hybridoma. Antibodies produced by this
approach only recognize a single epitope and they are all identical.
Georges Kohler
MONOCLONAL ANTIBODIES
Cesar Milstein
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Introduction to immunology. Lesson 6
Biotech applications – hybridomas and monoclonal antibodies
To make a hybridoma, we need the following:
Y
A well-purified antigen (even
better, just a single epitope) to
inject into lab animals along
with adjuvants
B-cells isolated from the
spleen of laboratory animals
immunized (injected) with the
antigen of interest
Commercially
available
myeloma/plasmacytoma cell
lines. These cells have to
fundamental features:
• They do not produce Ig of
their own
• They have a mutation in
the purines biosynthetic
pathway.
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Introduction to immunology. Lesson 6
Biotech applications – hybridomas and monoclonal antibodies
Purine synthesis occurs
trough 2 pathways
De Novo
Salvage
Aminopterin blocks
tetrahydrofolate production
Tetrahydrofolate
needed
Myeloma cells used for
hybridomas do not produce
HGPRT, so they use the de
novo pathway.
HypoxanthineGuanine
Phosphorybosiltransferase
(HGPRT) enzyme needed
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Introduction to immunology. Lesson 6
Biotech applications – hybridomas and monoclonal antibodies
In a medium containing aminopterin, HGPRT-null myeloma
cells are condemned to death because both the purine
pathways are blocked.
+
Y
B-cells can rescue these cells, because they express HGPRT
and can thus produce purines from hypoxanthine and
thymidine, in the absence of tetrahydrofolate.
Y
=
Hybridomas (fused cells) can survive in a medium with
Hypoxanthine, Aminopterin and Thymidine  HAT
medium
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Introduction to immunology. Lesson 6
Biotech applications – hybridomas and monoclonal antibodies
Monoclonal antibodies have thousands of
applications:
•
Research of tumor antigens
•
Research
of
specific
cell-types
markers
•
Identification of soluble molecules
and markers from biological samples
Abbas et al.
•
Diagnosis
•
Therapy
•
Etc.
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Introduction to immunology. Lesson 6
Biotech applications – applications of monoclonal antibodies: ELISA
Antibodies are very useful to “detect” soluble molecules in a liquid phase. A technique which
exploits this ability is called Enzyme-Linked Immunosorbent Assay (ELISA).
1
Antigens of interest can either be adsorbed to the bottom of
a culture plate (indirect ELISA) or captured by an antibody
adsorbed to the bottom of a culture plate (sandwich or
direct ELISA).
2
Another antibody is added to the mixture, which binds to
the antigen (detection antibody).
3
A final, modified antibody (secondary antibody) is added to
the mixture. This antibody binds to the detection antibody
and is covalently linked to a fluorochrome or to an enzyme
(typically HRP).
4
A chemical substrate is provided to the complex, and
metabolized by HRP to generate a colored product. The
more the color means the more the antibodies bound and,
hence, the more the quantity of the antigen!
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Introduction to immunology. Lesson 6
Biotech applications – applications of monoclonal antibodies: fluorescence
Antibodies are also very specific. So, you can use them to identify your cells of interest in a
mixture of different cells. The easiest way to identify these cells is to add a fluorescent moiety
to the antibody, and then look under a fluorescence microscope.
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Introduction to immunology. Lesson 6
Biotech applications – applications of monoclonal antibodies: humanized antibodies
It is not so difficult to generate antibodies against some markers (i.e., a tumor antigen) in lab
animals. But, if these antibodies are injected straightforward into humans, human B-cells will
recognize animal antibodies as non-self and produce antibodies against them (human antimouse antibodies, HAMAs), making them useless. Antibodies for therapy must be
humanized.
Murine
Humanized
Chimeric
Human
Humanizations consists in cloning the 3 CDR
regions of a mouse antibody against the
antigen of interest into a human Ig plasmid.
As a result, a new antibody is produced
which has the whole frame of a human
immunoglobulin but has the CDR (hence,
the antigen specificity) of the mouse
antibody.
Another possibility is to clone the whole VL
and VH region of a murine antibody into a
human antibody, creating a chimeric
antibody.
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Introduction to immunology. Lesson 6
Biotech applications – applications of monoclonal antibodies: humanized antibodies
Once humanized, antibodies can be used for therapy.
Examples of monoclonal antibodies for human therapy
Target
Effect
Disease
CD20
Depletion of B-cells
Rheumatoid
arthritis,
multiple sclerosis, other
autoimmune diseases
VEGF
Blockade of tumor neo- Breast cancer,
angiogenesis
carcinoma
HER2/Neu
Depletion of cells which
Breast cancer
show HER2 amplification
TNF
Inhibition
inflammation
Colon
of Rheumatoid
arthritis,
Crohn’s disease
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