Antibodies, Vaccines, Adjuants

Introduction to antibodies, vaccines,

adjuvants

Immunological agents

Biologics

In healthcare

management.

Polyclonal antibody

preparations induce passive

immunity

Vaccines active

immunization

Active immunization

Passive 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.

Immunize

large animaImmunize

large animal

(e.g. horse)

Collect

antibody-containing

antiserum/plasma

Initial purification

(precipitation)

High resolution

chromatographic

purification (e.g.

ion-exchange)

Centrifugation to clot: suspended

particles

Ethanol or

ammonium

sulphate

Production of antisera for therapeutic use:

Traditional polyclonal antibody preparation

Addition of

stabilizers,

preservatives and

adjustment of potency

Ion exchange chromatographic

technique

Sterile filtration and

aseptic filling

NaCl, glycine, phenol(

less than

0.25%)

Multidose

container filling

Liquid

productFreeze drying

Powdered

form

Sealed immediately in O2 free, N2 atmosph

ere

Prevent oxidativ

e degrada

tion during

storage

Antisera can induce unwanted side

effects

3.Purification, of

immunoglobulins from serum of

human donor is similar to of

antibodies from animals.

4.These may be purified from

donated blood of individuals who have recently:

5.been immunized against the antigen of interest;

6.recovered from an infection

caused by the antigen of interest.

These may generally be categorized into one of several groups upon the basis of their target

specificities.

These groups include antibodies raised

against:

1.specifi c microbial or viral pathogens;

2.microbial toxins;

3.snake/spider venoms (anti-venins).

The major polyclonal antibody preparations used therapeutically some are

• Anti-D immunoglobulin

• Botulism antitoxin

• Hepatitis A immunoglobulin

Basis of monoclonal antibody production

by hybridoma technology

Monoclonal antibody production

Screening:Phage display

technology

allows expression of exogenous (poly)peptides

phage particles.

genetic engineering of bacteriophages (viruses that infect

bacteria) and repeated rounds of

antigen-guided selection and phage

propagation.

biopanning

Tumour immunology

What is Tumor

immunology….?

Cancer immunology

is an interdisciplinary

branch of biology that

is concerned with

understanding the

role of the immune

system in the

progression and

development of

cancer.

The transformation of

a cell to the

cancerous state is

normally associated

with increased

surface expression of

antigens recognized

as foreign by the

host immune system.

Immunogenicity tumors Types of tumor antigens

found on tumor cells and only on

tumor cells

normal cells oncofetal Ag (usually

vrial induced)

Tumor

associated

antigens

Tumor specific

antigens

Immunosurveillance

TSA

Immune Sys

Recognize

Cell

Destroy

Anti tumour immune elements:

•

T lymphocytes

• recognizing

• lysing malignant cells

NK Cells

• Tumouricidal activity of NK

• cytokines (e.g. IL-2 and

Macrophages

• lysosomal enzymes

• reactive oxygen metabolites

Antibodies

• cell surface receptors

• bind to the antibody

Direct

By Toxic to the

cell

Indirect

Activation of

Immune

Elements

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.

Classes of Immunoglobulin's

IgM IgG

Isotypes

IgA IgD IgE

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),

Re Re

Immunoscintigraphy

• a γ-emitter

• technetium (99mTc)

• forming free sulfahydryl(…SH) group

• ascorbic acid or sodium dithioniteplanar gamma camera

Radioisotope

• carcinomas of the colon or rectum

• tomography scan or ultrasonography

• administered by injection

• nausea, fever, rash and headaches

CEA SCAN

Toxins conjugated to therapeutic

antibodies

calicheamicin

CD33 .Leukemia

Mylotarg

DownstreamSialic acid Fermentation

Immuno

sepression

Hematopoietic

Stem cell

DNA breakage

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

• Integral membrane glycoprotein.

• It is expressed mainly in the gut, liver and pancreas, gastrointestinal tract.

AFP

• Found in the circulatory system of the developing foetus.

• Detected in cancers of the liver, gastric and pancreatic cancer cells.

CA125

• That is expressed by up to 90 per cent of ovarian adenocarcinomas.

• Released from the tumor site into the general circulation.

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:

transformation by

Epstein- barr virus

infectionInduce low

cellular transformatio

n

Upon successful

transformation, produce low affinity

IgM antibodies

and cells are often

unstable

Fusion with murine

monoclonalsFusion of human

lymphocytes with murine

myeloma cells lead to

unstable hybrid.

Human genetic element loss is

observed.Common is the loss of

chromosome 2,14 and 22

Fusion with human

lymphoblastoid cell mines

Fusion of human

lymphocytes with human

lymphoblastoid cell lines is an inefficient

process

Problems related to human antibody

producing lymphocytes:-

this is possible but difficult as well.

unethical.

antigens administration to humans could endanger their health.

Although B-lymphocytes could be obtained from the peripheral circulation, the majority of these are unstimulated, and

impractical.

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 antibody is an antibody made by combining

genetic material from a

nonhuman source, like a mouse, with

genetic material from a human

being.

when used in therapeutic treatments,Chimeric antibodies are generally around two thirds human, reducing the risk of a reaction to foreign antibodies from a non-human animal.

A closely related concept is a

humanized antibody, made in a similar way but containing closer

to 90% human genetic material.

Chimaeric antibodies when compared with

murine antibodies, must be

Significantly less immunogenic

Display a prolonged serum

half life

Allow activation of various Fc

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

detection and treatment of cardiovascular disease, infectious agents, and various additional medical conditions.

Through Imaging monoclonals bacterial infections sites can be visualized. Radiolabelled antibodies can be used to achieve binding affinity for specific bacterial surface antigens.

1–2 per cent US population suffer from autoimmune conditions, like rheumatoid arthritis, MS and some forms of diabetes. Immunotherapeutic approach to treat such diseases is to induce depletion of T and B cells population.

additional use in transplantation-related medicine. In some instances, transplant patients must be maintained on immunosuppressive drugs (e.g. some steroids and, often, the fungal metabolite cyclosporine).

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

antigens found

in a sterile filtrate of cultures

of this

microorganism

Active immunization

against anthrax

BCG (bacillus Calmette–

Guérin) vaccine

Live attenuated strain of

Mycobacterium

tuberculosis

Active immunization

against

tuberculosis

Brucellosis vaccine Antigenic extract of Brucella

abortus

Active immunization

against brucellosis

Cholera vaccine Dead strain(s) of Vibrio

cholerae

Active immunization

against cholera

Cytomegalovirus vaccines Live attenuated strain of

human

cytomegalovirus

Active immunization

against

cytomegalovirus

Diphtheria vaccine Diphtheria toxoid formed by

treating

diphtheria toxin with

formaldehyde

Active immunization

against diphtheria

Japanese encephalitis

vaccine

Inactivated Japanese

encephalitis virus

Active immunization

against viral

agents causing Japanese

Categorization of vaccines:Attenuated, dead

or inactivated

bacteria

An attenuated

bacterial vaccine is

represented by BCG.

BCG is a strain

of tuberculae bacillus

that fails to cause

tuberculosis

o heat treatment;

o treatment with

formaldehyde or

acetone;

o treatment with

phenol or phenol

and heat;

o treatment with

Attenuated and

inactivated viral

vaccines

Many of the more

prominent vaccine

preparations in current

medical use consist of

attenuated

viral particles.Example

Mumps vaccines

consist of live

attenuated strains of

Paramyxovirus

parotitidis. In many

world regions,

routinely to vaccinate

childrenpart of a

combined

measles, mumps and

Toxoids and

antigen-based

vaccines

Diphtheria and tetanus

vaccines are two

commonly used

toxoid-based vaccine

preparations. The

initial stages of

diphtheria vaccine

production entail the

growth of

Corynebacterium

diphtheriae.

The toxoid is then

prepared by treating

the active toxin

produced with

formaldehyde. The

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 :

Production and quality control simpler.

It has fessibilityeven if virus

cannot be cultivated.

It is safer in cases

where are virus are oncogeni

c.

It has no NA or external

proteins therefore

establish a persistent 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 virusAssimilate

large quantity of DNA in its

genome.

Has ease of production.

Production is low cost.

Stability of freeze-dried

finished vaccine product.

As vaccine agent, prior history of

successful use.

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

developmentA 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 recombinantBaculovirus vector

Centrifugation andfiltration

Formulation as an oil-in water emulsion

β-propiolactone treatment

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

Safe

The preparation would elicit a protective

immune response with antigens.

promote an appropriate immune response, namely cellular or antibody

immunity depending on requirements for

protection

The adjuvant would be stable with regard to adjuvanticity and toxicity without any interaction with the

antigen.

It would be biodegradabl

e and immunologic

ally inert.

cheap to produce…..

..

…

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