Antibodies, Vaccines, Adjuants

Introduction to antibodies, vaccines,

adjuvants

Immunological In
agents healthcare
Biologics management.

Polyclonal
antibody Vaccines
preparations active
induce passive immunization
immunity
 Active Passive
immunization immunization
 Contents of antibodies
What are antibodies

Traditional polyclonal antibody preparations

Monoclonal antibodies

Screening of monoclonal antibodies

Tumour immunology

Immunosurveillance

Antibody structure

Antibody-based strategies for tumour detection/destruction

Immunoscintigraphy
Drug-based tumor immunotherapy

ADEPT

The etoposide–alkaline phosphatase ADEPT system

First-generation anti-tumour antibodies: clinical disappointment

Tumor-associated antigens

Antigenicity for murine monoclonals

Problems related to human antibody producing lymphocytes

Chimaeric and humanized antibody

AVASTIN=Bevacizumab

therapeutic applications of antibodies

Antibodies
 Antibody (immunoglobulin)
 protective protein immune
system antigen.

Monoclonal antibodies: Derived from a single B

cell clone
 Monospecific

Polyclonal antibodies: collection of

antibodies from different B cells
that recognize multiple epitopes on
the same antigen.
Production of antisera for therapeutic use:
Traditional polyclonal antibody preparation
Immunize
Immunize
large
largeanima
animal
(e.g. horse)

Collect
antibody-containing
antiserum/plasma

Ethanol
Centrifugati
Initial purification or
on to clot:
(precipitation) ammoniu
suspended
m
particles
sulphate

High resolution
chromatographic
purification (e.g.
ion-exchange)
 Ion
exchange
chromato
graphic
technique
Addition of
stabilizers,
preservatives and
adjustment of potency NaCl,
glycine, Multido
phenol( se
less contain
Prevent than er filling
Sealed Sterile filtration and 0.25%)
immediat oxidativ
ely in O2 e aseptic filling
free, N2 degrada
atmosph tion
ere during
storage
Liquid
Freeze drying
product

Powdered
form
Antisera can induce unwanted side
effects

6.recovered from
an infection
caused by the
antigen of
interest.
3.Purification,
of
immunoglobu
5.been lins from
immunized 4.These may be
purified from serum of
against the
antigen of donated blood of human donor
interest; individuals who is similar to of
have recently: antibodies
from animals.
 These may generally
be categorized into one
of several groups upon The major
the basis of their target polyclonal antibody
specificities.
preparations used
These groups include therapeutically
antibodies raised
against: some are
1.specifi c microbial or • Anti-D
viral pathogens; immunoglobulin
2.microbial toxins; • Botulism antitoxin
• Hepatitis A
3.snake/spider venoms immunoglobulin
(anti-venins).
Basis of monoclonal antibody production
by hybridoma technology
Monoclonal antibody production
 Screening:Phage display
technology

genetic engineering
of bacteriophages
allows expression (viruses that infect
of exogenous bacteria) and
(poly)peptides repeated rounds of
phage particles. antigen-guided
selection and phage
propagation.
biopanning
Tumour immunology
What is Tumor
immunology….?

Cancer immunology
The transformation of
is an interdisciplinary
a cell to the
branch of biology that
cancerous state is
is concerned with
normally associated
understanding the
with increased
role of the immune
surface expression of
system in the
antigens recognized
progression and
as foreign by the
development of
host immune system.
cancer.
Immunogenicity tumors
Types of tumor antigens

Tumor
Tumor specific
associated
antigens
antigens

found on tumor cells and only on

tumor cells
normal cells oncofetal Ag (usually
vrial induced)
Immunosurveillance

TSA

Immune Sys
Recognize

Cell
Destroy
 Anti tumour immune elements:
•

T lymphocytes NK Cells Macrophages Antibodies

• recognizi • Tumouri • lysosom • cell

ng cidal al surface
• lysing activity enzymes
Direct
receptor
Indirect
malignan of NKBy Toxic to•the
reactive s of
Activation
Immune
t cells • cytokine cell oxygen • bind to
Elements
s (e.g. metabolit the
 Classes of Immunoglobulin's

IgM IgG IgA IgD IgE

Isotypes

Human Murine
IgG1, IgG2, IgG3 and IgG4 IgG1, IgG2a, IgG2b and
IgG3
 rDNA tehnolgy : FV domains produced and stabilized by
 covalent linkage
 Single-chain
 ‘hypervariable’ : greater variability in amino acid sequence
than other variable regions. This is also called (CDRs).
 framework regions.
 Igs are glycoproteins.
Antibody Architecture
 Antibody-based strategies for tumour
detection/destruction Clear

 Binding of unaltered monoclonal antibodies to a

tumour surface alone should facilitate increased
destruction of tumour cells.
 radioactive tag conjugate anti cancer
agent
β-emitters: medium energy, penetrating a
thickness of several cells.
 Radioisotopes:
killing all cells
α-emitters: Higher energy, only about one
cell deep.
Examples:
(125I, 131I),
 Immunoscintigraphy

• a γ-emitter
• technetium (99mTc)
Radioisotope • forming free sulfahydryl
(…SH) group
• ascorbic acid or sodium
dithioniteplanar gamma
camera
• carcinomas of the colon or rectum
• tomography scan or
CEA SCAN ultrasonography
• administered by injection
• nausea, fever, rash and headaches
 Toxins conjugated to therapeutic
antibodies

caliche CD33
Mylotarg amicin .Leukemia

Sialic acid Downstream Fermentation

Immuno
DNA Hematopoietic
breakage sepress Stem cell
ion
  Drug-based tumor immunotherapy
 ADEPT
The etoposide–alkaline phosphatase ADEPT system
First-generation anti-tumour antibodies: clinical
disappointment
 Tumor-associated antigens
Drug-based tumor
immunotherapy
 This involves conjugation of a chemotherapeutic
drug to a tumor-specific antibody.
 Drugs adriamycinand and methotrexate.
 A limited number of drug molecules can be
conjugated to each antibody molecule.
Antibody-directed enzyme prodrug
therapy (ADEPT)

 An enzyme conjugated to an antitumor antibody

is given and localizes in the tumor. A prodrug is
then given, which is converted to a cytotoxic drug
selectively in the tumor.
 A single antibody–enzyme conjugate would
activate many molecules of the prodrug in
question.
ETOPOSIDE
 Etoposide is administered in prodrug
 A semi-synthetic derivative of podophyllotoxin.
 It is used as an anti-cancer agent. Its cellular
uptake is diffusion dependent, and once inside
the cell it exerts its cytocidal effect.
First-generation anti-tumor
antibodies: clinical disappointment
 Insufficient information
 Murine monoclonals prompt an immune response
when administered to humans
 Poor penetration of tumor mass by antibody
 Short half-life when administered to humans
Tumor-associated antigens
 Tumor assosiated antigen represents any antigen
associated with any cancer cell.
 Many tumor types are induced by chemical
and/or physical.
 Carcinogen induces a point mutation in a
nucleotide sequence altering expression levels of
the gene .
Conti…
 DNA viruses, such as adenoviruses induce
cellular transformation in rodents.
 Other viruses have been implicated in human
cancers.
 Certain RNA viruses, retroviruses are capable of
inducing cancer.
Conti..
 Another group is oncofoetal antigens.
 These antigens are proteins
 Expressed during certain stages of foetal
development.
 Oncofoetal antigens represent important potential
diagnostic markers. CEA, AFP and CA125
 CEA AFP CA125

• Integral membrane • Found in the • That is expressed

glycoprotein. circulatory system by up to 90 per
• It is expressed of the developing cent of ovarian
mainly in the gut, foetus. adenocarcinomas.
liver and pancreas, • Detected in • Released from the
gastrointestinal cancers of the liver, tumor site into the
tract. gastric and general circulation.
pancreatic cancer
cells.
Antigenicity for murine monoclonals
 Inherent therapeutic limitations that associate
with administration of murine monoclonals.
 HAMA can be detected within the 14 days of
antibody administration.
 Repeated administration will increase the HAMA
response significantly.
 But HAMA response will destroy subsequent
monoclonal doses administered.
 overcoming the immunogenicity problem would
be the generation and use of monoclonal
antibodies of human origin.
 Human antibody producing lymphocytes can
potentially be done immortal by:
transformati
on by Fusion with
Fusion with
human
Epstein- barr murine
lymphoblastoid
virus
Induce low monoclonals
Fusion of
cell mines
cellular
infection human
transformatio lymphocytes
n with murine Fusion of
Upon myeloma cells human
lead to lymphocytes
successful unstable
transformatio hybrid. with human
n, produce Human genetic lymphoblastoi
low affinity element loss is d cell lines is
IgM observed.Comm an inefficient
on is the loss of process
antibodies chromosome
and cells are 2,14 and 22
often
unstable
Problems related to human antibody
producing lymphocytes:-
Although
B-
antigens lymphocyt
es could
administ be
this is
ration to obtained
possible
unethica humans from the impracti
but peripheral
l. could cal.
difficult circulation
endang
as well. , the
er their majority of
health. these are
unstimulat
ed, and
Chimaeric and humanized
antibody

Recombinant DNA
technology has
provided an alternative
and successful route
of reducing the innate
immunity of murine
monoclonals.
What are Chimaeric antibodies??
A Chimaeric
when used in
antibody is an therapeutic
antibody made treatments,Chi
by combining meric antibodies
genetic material are generally A closely related
around two concept is a
from a humanized antibody,
thirds human,
nonhuman reducing the
made in a similar way
source, like a but containing closer
risk of a to 90% human
mouse, with reaction to genetic material.
genetic material foreign
from a human antibodies from
a non-human
being. animal.
 Chimaeric
antibodies when
compared with
murine antibodies,
must be

Display a Allow activation of

Significantly less
prolonged serum various Fc
immunogenic
half life mediated functions
 In practice, the expected reduced immunogenicity
was observed.
 clinical trials with Chimaeric are generally safe
and non-toxic.
 The immune responses rate observed after single
dose administration dropped from almost 80 per
cent (murine) to in the region of 5 per cent
(Chimaeric).
What is humanization??
Humanization (reshaping and CDR-
grafting ) is a well established technique
to reduce the immunogenicity of mAbs
from xenogenic sources

The mechanics to engineer mAbs by

molecular biology technique is
straightforward

Simple grafting of the rodent CDRs into

human framework not always reconstruct
the binding affinity and specificity of the
original mAbs
Product case study;
AVASTIN=Bevacizumab
 149 kDA rec. humanized antibody developed by
GENETECh
 Firstly approved for medical use in USA(2004)
and Europe(2005)
 Treat colorectal cancer patients with
chemotherapeutic drugs
 Inhibit angiogenesis (a process require tumor
growth)
 Purified by protein A affinity chromatography
 Fatal side effects like gastrointestinal perforation,
wound healing complications and hemorrhage
Antibody fragments

Fragments(ab),F (ab)2,Fv easily

generated
• Used for diagnostic and therapeutic
purpose
• Effective due to Low molecular mass
• If Chimaeric/ humanized Ab,effective
as therapeutic
• Radiolabelled fragments better suited to
diagnostic imaging purpose
therapeutic applications
Through Imaging
monoclonals bacterial
detection and infections sites can be
treatment of visualized.
cardiovascular Radiolabelled
disease, infectious antibodies can be
agents, and various used to achieve
additional medical binding affinity for
1–2conditions
per cent .US
population suffer from specific
additional bacterial
use in
autoimmune conditions, surface antigens.
transplantation-related
like rheumatoid arthritis, medicine. In some instances,
MS and some forms of transplant patients must be
diabetes. maintained on
Immunotherapeutic immunosuppressive drugs
approach to treat such (e.g. some steroids and, often,
diseases is to induce the fungal metabolite
depletion of T and B cells cyclosporine).
population.
Vaccine Technology
 Contents in vaccine technology
Intro about vaccine technology

Traditional vaccine preparation

Categorization of vaccines

Hbs based vaccines

Impact of genetic engineering

Peptide vaccines

Merits of peptide vaccines

Treatment of diseases by peptide vaccines

Vaccine vectors

Development of AIDS vaccine

Difficulties associated with vaccine development

AIDS vaccine in clinical trials

Vaccine Technology

 Fundamental element  Modern medicinal approach.

 Preventive approaches Infectious diseases.
 The global vaccine market US$3 billion.
Continue…..

Multinational immunization program

500 000 adults die annually in the USA.

Due to pneumococcal pneumonia, influenza and hepatitis B.

A Vaccine is mixture of antigenic components that are

derived from or related to a pathogen.
In most cases upon vaccine administration both the humoral
and cell- mediated responses are activated.
Traditional Vaccine Preparations:

• Vaccines that are developed through Recombinant

DNA technology.
• Approximately 30 vaccines out of which few are
listed in a table below .
Product Description Application
Anthrax vaccines Bacillus anthracis-derived Active immunization
antigens found against anthrax
in a sterile filtrate of cultures
of this
microorganism
BCG (bacillus Calmette– Live attenuated strain of Active immunization
Guérin) vaccine Mycobacterium against
tuberculosis tuberculosis
Brucellosis vaccine Antigenic extract of Brucella Active immunization
abortus against brucellosis
Cholera vaccine Dead strain(s) of Vibrio Active immunization
cholerae against cholera
Cytomegalovirus vaccines Live attenuated strain of Active immunization
human against
cytomegalovirus cytomegalovirus
Diphtheria vaccine Diphtheria toxoid formed by Active immunization
treating against diphtheria
diphtheria toxin with
formaldehyde
Japanese encephalitis Inactivated Japanese Active immunization
vaccine encephalitis virus against viral
 Categorization of vaccines:
Attenuated, dead Attenuated and Toxoids and
or inactivated inactivated viral antigen-based
bacteria vaccines vaccines
An attenuated Many of the more Diphtheria and tetanus
bacterial vaccine is prominent vaccine vaccines are two
represented by BCG. preparations in current commonly used
BCG is a strain medical use consist of toxoid-based vaccine
of tuberculae bacillus attenuated preparations. The
that fails to cause viral particles.Example initial stages of
tuberculosis Mumps vaccines diphtheria vaccine
consist of live production entail the
o heat treatment; attenuated strains of growth of
o treatment with Paramyxovirus Corynebacterium
formaldehyde or parotitidis. In many diphtheriae.
acetone; world regions, The toxoid is then
o treatment with routinely to vaccinate prepared by treating
phenol or phenol childrenpart of a the active toxin
and heat; combined produced with
HBs Ag based vaccines:
Impact of Genetic Engineering:

Production of a clinically
safe product.

Production of subunit vaccine

in an unlimited supply.

Consistent production of a
defined product that would
thus be less likely to cause
unexpected side effects
Peptide vaccines
Peptide vaccines
A peptide vaccine is any peptide which
serves to immunize an organism

against a pathogen. Peptide vaccine are

often synthetic and mimic naturally
occurring proteins from pathogens

They are stable in cheap and

manufacturing

Less quality assurance is needed

Advantages :

It has It is safer It has no NA

Productio or external
fessibility in cases
n and proteins
even if where are
quality therefore
virus virus are establish a
control
cannot be oncogeni persistent
simpler.
cultivated. c. infection
Treatment:
Peptide vaccines are used to treat many diseases
which are in the following :
 Synthetic peptides are not applicable to all
viruses. In case of poliovirus, this approach did
not work because two or more different viral
capsid proteins made important anti-genetic sites.
 In foot and mouth disease , protection is
achieved by immunizing animals with linear
sequence of 20 amino acids .
 Forty plasmodium antigens have been identified
and they help in formation of anti-malaria
vaccines targeted to various stages in parasite
life cycle .
Vaccine vectors:
 The strategy of vaccine vectors involves
incorporation of DNA coding for pathogen-
derived antigen into a non-pathogenic species.
If it express the gene then it may be used to
immunize against the pathogens of interest.
 In general recombinant viral vectors elicit both
strong humoral and cell-mediated immunity
.The immunological response is less
pronounced.
Factors render vector system :
vaccinia virus
Assimilate
large quantity
As vaccine of DNA in its
agent, prior genome.
history of
successful Has ease of
use. production.

Stability of
freeze-dried Production
finished is low cost.
vaccine
product.
Examples
1.Poxvirus:

They are large , enveloped double stranded DNA viuses. The most studied
virus of this family are variola and vaccinia

2. Adenovirus:

They are double stranded DNA virus

They can prompt respiratory tract infections

Like adenovirus strains that cause asymptomatic infection and which have
proven to be very safe and effective adenovirus vaccines has been isolated .
DEVELOPMENT OF AIDS
VACCINE
AIDS was initially described in the U.S in
1981.
By 1983,the causative agent,now termed
HIV,was identified.
Member of lentivirus subfamily of
retroviruses.
Spherical,enveloped particle,100-150nm in
diameter.
Contain RNA as its genetic material.
Preventive HIV vaccine
 a vaccine designed to prevent
getting infected from HIV.
 Cellular system:
Therapeutic HIV vaccine:
 a vaccine designed to boost
the immune response to HIV in
a person already infected with
the virus (immune based
therapy). Also referred to as an
immunotherapeutic vaccine
Preventive HIV vaccine
 a vaccine designed to prevent
getting infected from HIV.
 Cellular system:
Therapeutic HIV vaccine:
 a vaccine designed to boost
the immune response to HIV in
a person already infected with
the virus (immune based
therapy). Also referred to as an
immunotherapeutic vaccine
Attachment and entry
 In excess of 40 million individuals are now thought to
be infected by HIV. In 2001 alone, it was estimated
that 3 million people died from AIDS and a further 5
million became infected with the virus.
 Over 20 million people in total are now thought to
have died from AIDS.
 The worst affected geographical region is the
southern half of Africa.
 So far, no effective therapy has been discovered, and
the main hope of eradicating this disease lies with the
development of safe, effective vaccines.
Difficulties associated with vaccine
development
A number of attributes of HIV and its mode of infection
conspire to render development of an effective vaccine.
These factors include:
HIV displays extensive genetic variation even within a
single individual.

HIV infects and destroys T-helper lymphocytes.

Although infected individuals display a wide range of
antiviral immunological responses, these ultimately fail
to destroy the virus.
The infection may often be spread not via transmission
of free viral particles, but via direct transmission of
infected cells.
AIDS vaccines in clinical trials

 monomeric HIV-1 Env gp120 protein, and the

aim of this strategy was to induce Env-specific
humoral immune responses.
 In early-phase clinical trials, gp120
immunogens elicited type-specific binding
antibodies but failed to induce broadly reactive
neutralizing antibodies
 The second vaccine concept that has completed
clinical efficacy studies involved replication-
incompetent recombinant adenovirus serotype 5
(rAd5) vectors expressing HIV-1 Gag, Pol and Nef .
 The aim of this strategy was to elicit HIV-1-specific
cellular immune responses
 Another phase 3 study evaluating the efficacy of a
recombinant canarypox vector prime/gp120 protein
boost vaccine regimen is currently underway
Cancer vaccines

 The aim of cancer vaccines is to stimulate the

immune system to be able to recognise cancer cells
as abnormal and destroy them.
 Each type of cancer vaccine works on the same
basic idea that the vaccine, which contains tumor
cells or antigens, stimulates the patient's immune
system, which produces special cells that kill cancer
cells and prevent relapses of the cancer.
 Majority will probably be used as adjuvants.
 Researchers are actively trying to overcome hurdles
in the making of these vaccines
AIDS vaccines in clinical trials

 monomeric HIV-1 Env gp120 protein,

and the aim of this strategy was to
induce Env-specific humoral immune
responses. In early-phase clinical trials,
gp120 immunogens elicited type-
specific binding antibodies but failed to
induce broadly reactive neutralizing
antibodies
 The second vaccine concept that has completed
clinical efficacy studies involved replication-
incompetent recombinant adenovirus serotype 5
(rAd5) vectors expressing HIV-1 Gag, Pol and
Nef . The aim of this strategy was to elicit HIV-1-
specific cellular immune responses
 Another phase 3 study evaluating the efficacy of
a recombinant canarypox vector prime/gp120
protein boost vaccine regimen is currently
underway
Cancer vaccines
 The aim of cancer vaccines is to stimulate the
immune system to be able to recognise cancer
cells as abnormal and destroy them.
 Each type of cancer vaccine works on the same
basic idea that the vaccine, which contains tumor
cells or antigens, stimulates the patient's immune
system, which produces special cells that kill
cancer cells and prevent relapses of the cancer.
 Majority will probably be used as adjuvants.
 Researchers are actively trying to overcome
hurdles in the making of these vaccines
Recombinant veterinary vaccines

Fermentation of Spodopter frugiperda cells

Infection with recombinant

Baculovirus vector

Centrifugation and
filtration

β-propiolactone treatment

Formulation as an oil-in water emulsion

Adjuvants
INTRODUCTION
 An adjuvant is defined as any material that
enhances the cellular and/or humoral immune
response to an antigen
 .
 A vaccine adjuvant is a component that
potentiates the immune response to an antigen.

 The greater the product’s adjuvanticity, the more

toxic it is likely to be.
Properties of an ideal adjuvant
The preparation would
Safe elicit a protective
immune response with
antigens.
promote an
appropriate immune
response, namely
cellular or antibody
immunity depending
on requirements for
protection
cheap to
produce The adjuvant would
….. It would be be stable with regard
biodegradabl to adjuvanticity and
.. e and toxicity without any
…
immunologic interaction with the
ally inert
. antigen.
USES OF ADJUVANTS
Adjuvants have been used for high and long-lasting immune
response.

Adjuvants are very important for purified, synthetic vaccines which

are poorly immunogenic.

To reduce the amount of antigen or the number of immunizations

needed for protective immunity

Synthetic and subunit vaccines will be expensive to produce. With

the use of adjuvants, less antigen may be required to stimulate the
immune response, thus saving cost of vaccines
As antigen delivery systems for the uptake of antigens by the
mucosa.
Mechanisms of adjuvant action
 Adjuvants may exert their immune-enhancing effects according to the
following immune-functional activities.
 Vaccines are not enough to develop immune memmory
cells.
 Adding an adjuvants triggers the immune system become
more sensitive to vaccine.
 many immune cells come to ijection site,respond to
vaccine and become immune mammory cells.
 Latter if the original pathogen shows up,immune cells
remember and ready to attack.
Summary of adjuvants approved
for human use