Antibodies, vaccines, adjuvents

1. Antibodies, Vaccines, Adjuants

2. Introduction to antibodies, vaccines,
adjuvants
Immunological
agents
Biologics
In
healthcare
management.
Polyclonal
antibody
preparations
induce passive
immunity
Vaccines
active
immunization

3. Active
immunization
Passive
immunization

4. 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

5. 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

6. 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.

7. Immunize
large anima
Immunize
large animal
(e.g. horse)
Collect
antibody-containing
antiserum/plasma
Initial purification
(precipitation)
High resolution
chromatographic
purification (e.g.
ion-exchange)
Centrifugati
on to clot:
suspended
particles
Ethanol
or
ammoniu
m
sulphate
Production of antisera for therapeutic use:
Traditional polyclonal antibody preparation

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

9. Antisera can induce unwanted side
effects
3.Purification,
of
immunoglobu
lins 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.

10. 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

11. Basis of monoclonal antibody production
by hybridoma technology

12. Monoclonal antibody production

13. 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.

14. biopanning

15. Tumour immunology

16. 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.

17. 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

18. Immunosurveillance
TSA
Immune Sys
Recognize
Cell
Destroy

19. Anti tumour immune elements:
•
T lymphocytes
• recognizi
ng
• lysing
malignan
t cells
NK Cells
• Tumouri
cidal
activity
of NK
• cytokine
s (e.g.
Macrophages
• lysosom
al
enzymes
• reactive
oxygen
metabolit
Antibodies
• cell
surface
receptor
s
• bind to
the
Direct
By Toxic to the
cell
Indirect
Activation of
Immune
Elements

21. 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

22. Antibody Architecture

23. 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),

24. 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

25. Toxins conjugated to therapeutic
antibodies
caliche
amicin
CD33
.LeukemiaMylotarg
DownstreamSialic acid Fermentation
Immuno
sepress
ion
Hematopoietic
Stem cell
DNA
breakage

26.  Drug-based tumor immunotherapy
 ADEPT
The etoposide–alkaline phosphatase ADEPT system
First-generation anti-tumour antibodies: clinical
disappointment
 Tumor-associated antigens

27. 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.

28. 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.

30. 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.

32. 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

33. 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 .

34. 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.

35. 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

36. 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.

37. Antigenicity for murine monoclonals

38.  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.

39.  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:

40. transformati
on by
Epstein- barr
virus
infection
Induce low
cellular
transformatio
n
Upon
successful
transformatio
n, produce
low affinity
IgM
antibodies
and cells are
often
unstable
Fusion with
murine
monoclonals
Fusion of
human
lymphocytes
with murine
myeloma cells
lead to
unstable
hybrid.
Human genetic
element loss is
observed.Comm
on is the loss of
chromosome
2,14 and 22
Fusion with
human
lymphoblastoid
cell mines
Fusion of
human
lymphocytes
with human
lymphoblastoi
d cell lines is
an inefficient
process

41. Problems related to human antibody
producing lymphocytes:-
this is
possible
but
difficult
as well.
unethica
l.
antigens
administ
ration to
humans
could
endang
er their
health.
Although
B-
lymphocyt
es could
be
obtained
from the
peripheral
circulation
, the
majority of
these are
unstimulat
ed, and
impracti
cal.

42. Chimaeric and humanized
antibody
Recombinant DNA
technology has
provided an alternative
and successful route
of reducing the innate
immunity of murine
monoclonals.

43. 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,Chi
meric 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.

44. 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

45.  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).

46. 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

47. 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

48. 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

49. 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).

50. Vaccine Technology

51. 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

52. Vaccine Technology
 Fundamental element  Modern medicinal approach.
 Preventive approaches Infectious diseases.
 The global vaccine market US$3 billion.

53. 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.

54. Traditional Vaccine Preparations:
• Vaccines that are developed through Recombinant
DNA technology.
• Approximately 30 vaccines out of which few are
listed in a table below .

55. 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

56. 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;
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
childrenpart of a
combined
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

57. HBs Ag based vaccines:

58. 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

59. Peptide vaccines

60. 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

61. Advantages :
Productio
n and
quality
control
simpler.
It has
fessibility
even 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

62. 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 .

63. 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.

64. Factors render vector system :
vaccinia virus
Assimilate
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.

65. 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 .

66. DEVELOPMENT OF AIDS
VACCINE

67. 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.

68. 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

69. 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

70. Attachment and entry

72.  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.

73. 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.

74. 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

75.  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

76. 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

77. 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

78.  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

79. 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

80. Recombinant veterinary vaccines
Fermentation of Spodopter frugiperda cells
Infection with recombinant
Baculovirus vector
Centrifugation and
filtration
Formulation as an oil-in water emulsion
β-propiolactone treatment

81. Adjuvants

82. 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.

83. 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
…..
..
…

84. 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.

85. 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.

86. Summary of adjuvants approved
for human use