4. OUTLINE
Introduction to vaccines
Modes of Immunization
Types of Vaccines
Production of Vaccine by rDNA techniques
Adjuvants, Vaccine Delivery Systems, and ideal
vaccine
Vaccine Storage and Handling.
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5. Introduction to vaccines
o Vaccine is immunological preparations.
o It directs the immune system toward a particular disease in a
specific manner.
o Employ recombinant techniques to create fractions of a
microorganism or virus that retain the activity as an immune
stimulant for protection against that infectious species;
Hepatitis B vaccines were the first recombinant vaccines
created for immunization.
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6. o Vaccination (Immunization):
is a procedure designed to
increase concentrations of
antibodies and/or effector
T-cells which are reactive
against infection.
Intro…….
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7. Modes of Immunization
1. Active Immunization:
o Involves the inoculation of immunogenic materials
(antigens) to induce an immune response (and therefore
immunological memory) in the recipients.
o It takes longer to develop than passive but lasts much
longer, and may often be life-long.
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8. Mods of immunization…..
o In active immunization, various forms of antigenic
material may serve as a vaccine:
Killed organisms
Attenuated organisms
Toxoids
Purified antigens, and
“Naked” DNA and etc.
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9. Mods of immunization…..
2. Passive immunization:
o Achieved by giving immunoglobulins and the
protection is immediate but lasts only a few weeks.
o Injection of antibody to a pathogen can provide very
rapid, short-lived, resistance to infection;
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10. Mods of immunization…..
10
o Generally used when:
There is no time to wait for the development of active
immunity, or
There is no effective active vaccine exists.
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11. Vaccine used in passive immunization…..
Human antibodies
o Normal human IgG, prepared from individual
donors, contains significant levels of antibody to
measles and hepatitis viruses;
o Protective antibody for tetanus can be obtained from
immunized donors;
o Anti-Zoster antibody collected from patients during
recovery from an infection.
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12. Vaccine used in passive immunization…..
Heterologous antibodies
Horse antibodies to diphtheria toxin or toxins of snake
and spider venoms have been very effective in
neutralizing the effect of these dangerous molecules.
The use of heterologous serum, of course, introduces a
substantial risk of inducing serum sickness or an
allergy.
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15. 1. Killed pathogen vaccine
The viruses which cause rabies, influenza and polio
can be collected, killed by treatment with heat or
chemicals, and used as effective vaccinating agents;
The bacteria responsible for cholera (Vibrio
cholerae), pertussis (Bordetella pertussis) and
typhoid fever (Salmonella typhi) can be used.
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16. Killed pathogen vaccine……
Inactivated whole virus vaccines are the easiest preparations to use:
The outer virion coat should be left intact but the replicative
function should be destroyed.
To be effective, non-replicating virus vaccines must contain
much more antigen than live vaccines that are able to
replicate in the host.
The chemicals used include formaldehyde or
betapropiolactone.
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17. Killed pathogen vaccine……
o Excessive treatment can destroy immunogenicity
whereas insufficient treatment can leave infectious
virus capable of causing disease.
o Potential safety problems of killed vaccines:
Incomplete inactivation
Increased risk of allergic reactions due to large
amounts of antigen involved.
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18. 2. Attenuated vaccine
o Live virus vaccines are prepared from attenuated strains
that are almost or completely devoid of pathogenicity but
are capable of inducing a protective immune response.
Live viruses, but in a weakened or "attenuated" form,
provide effective vaccination for measles, mumps, polio
and influenza.
Attenuated bacterial vaccines for anthrax and for
tuberculosis (BCG, “Bacille de Calmette-Guerin”, an
attenuated form of the organism which causes bovine
tuberculosis). 8/13/2022
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19. Attenuated vaccine….
Attenuation of viruses may be achieved by growth
in vitro in the laboratory and selection of genetic
variants with limited pathogenic potential
Attenuation of bacteria may be achieved by
allowing bacterial cultures to "age" during laboratory
culture with or without deliberate selection of
variants.
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20. Attenuated vaccine….
o Potential safety problems of live vaccines:
Under-attenuation;
Mutation leading to reversion to virulence;
Preparation instability;
Contaminating viruses in cultured cells,
Heat lability;
Should not be given to immunocompromised or pregnant
patients. 8/13/2022
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21. 3. Purified antigens
Toxoids
o In the case of diphtheria and tetanus infections, the
real danger in the disease comes not from the
presence of the organisms themselves but from the
potent toxins which they produce.
o Effective immunity can be induced by immunization
with chemically modified toxins(toxoids), which are
no longer toxic but still highly immunogenic.
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22. Purified antigens…..
o Originally, non-replicating vaccines were derived from crude
preparations of virus from animal tissues.
o It is now possible to identify the peptide sites encompassing
the major antigenic sites of viral antigens, from which highly
purified subunit vaccines can be produced.
o Increasing purification may lead to loss of immunogenicity.
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23. Purified antigens…..
o Examples of purified subunit vaccines include
Vaccines for meningococcus, pneumococcus, the HA
vaccines for influenza A and B, and HBsAg;
“Influenza HA Vaccine” is a clear or slightly whitish
turbid liquid product containing hem-agglutinin
(referred to as "HA" in monograph) of influenza virus.
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24. Purified antigens…..
o Advantage over attenuated vaccines is not posing any
risk of infection, and over killed organism vaccines is
being less likely to cause severe inflammatory reactions.
o Disadvantages:
May be less immunogenic than conventional inactivated
whole-virus vaccines
Requires adjuvant
Requires primary course of injections followed by boosters
Fails to elicit CMI (cell-mediated immune responses).
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25. Limitations to Traditional Vaccines
26
All organisms can’t grow in culture
Safety to lab personnel and expense
Insufficient attenuation and reversion to
infectious state
Need refrigeration
Do not work for all infectious agents;
Infants/children receive them – immature
immunity.
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26. Quiz (5%)
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27
1. Write the difference between innate and acquired
immunity in detail (2%).
2. What is the difference between killed vaccine and life
attenuated vaccine and give example for each (2%).
3. Write limitations of traditional type of vaccine (1%).
28. 1. rDNA Vaccine
o Altered Plasmid DNA encoding the desired antigen
is administered parenterally.
o The foreign protein is expressed by the host cell and
generate an immune response.
o In theory these vaccines would be extremely safe
and devoid of side effects since the foreign antigens
would be directly produced by the host animal.
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29. rDNA Vaccine
o Involves the direct injection of
plasmids - loops of DNA that
contain genes for proteins
produced by the organism being
targeted for immunity.
then DNA is taken up by host
cells, which then start
expressing the protein
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30. Advantages Disadvantages
Low intrinsic immunogenicity Unknown effect of long-term
expression
Induction of long-term immunity Formation of anti-DNA antibodies
yield a strong cell-mediated response
without using of live virus vaccines
Induction of both humoral and cellular
responses
Possible genome integration
Possibility of constructing multiple-
epitope plasmid
Heat stability
Ease of large-scale production 8/13/2022
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rDNA Vaccine……
31. 2. Recombinant Protein Vaccine
o Incorporation of the
gene responsible
peptide sequence into
a plasmid and
peptides expressed in
host cells.
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32. 3. Recombinant Vector Vaccine
o There are several drawbacks in traditional strategy of
attenuation:
The virulence and life cycle of the pathogen must be
known in detail;
Traditional attenuation may result in reduced
immunogenicity
Reversion of attenuated microorganism is possible
either during its production or presence in the host.
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33. Recombinant Vector Vaccine…..
Reassortment and Reverse Genetics are important
processes used in production of Recombinant Vector
Vaccines:
Reassortment:
The swapping and mixing of gene segments between
two different viral strains inside a fertilized chicken egg,
to create a new vaccine.
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36. 4. Virus-Like Particles (VLPs)
• It is comprised of multiple
copies of protein antigens
that when assembled
together mimic the
appearance of a virus.
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37. Production of vaccines by rDNA Techniques
o Recombinant DNA technology is being used to
produce a new generation of vaccines;
Virulence genes are deleted and organism is still able
to stimulate an immune response.
If the agent cannot be maintained in culture, genes of
proteins for antigenic determinants can be cloned and
expressed in an alternative host; e.g. E. coli.
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38. Production by rDNA techniques…
Recombinant viral proteins
o Virus proteins are expressed in bacteria, yeast and
mammalian cells
o E.Coli cells were first to be used for this purpose, but
the expressed proteins were not glycosylated;
o Recombinant hepatitis B vaccine is the only
recombinant vaccine licensed at present.
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39. Production by rDNA techniques…
o An alternative application of recombinant DNA technology is
the production of hybrid virus vaccines.
The best known example is vaccinia; the DNA sequence
coding for the foreign gene is inserted into the plasmid vector
along with a vaccinia virus promoter and vaccinia thymidine
kinase sequences.
The resultant recombination vector is then introduced into
cells infected with vaccinia virus to generate a virus that
expresses the foreign gene.
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40. Production by rDNA techniques…
The recombinant virus vaccine can then multiply in infected cells
and produce the antigens of a wide range of viruses.
The genes of several viruses can be inserted, so the potential exists
for producing polyvalent live vaccines.
HBsAg, rabies, HSV and other viruses have been expressed in
vaccinia.
Hybrid virus vaccines are stable and stimulate both cellular and
humoral immunity.
They are relatively cheap and simple to produce.
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42. o Chemicals in the vaccine solution that enhance the immune
response;
1. Functions to localize and slowly release antigen at or near
the site of administration.
2. Functions to activate APCs (antigen presentating cells) to
achieve effective antigen processing or presentation
E.g. Alum – Ag is the vaccine clumps with the alum such that
the Ag is released slowly gives more time for memory cells to
form.
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Adjuvant….
43. Adjuvant….
o Materials that have been used include:
1. Aluminium salts
2. Mineral oils
3. Mycobacterial products; e.g. Freund's adjuvants.
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44. Adjuvant….
o The most effective experimental adjuvant known,
Freund's Adjuvant, cannot be used in clinical
medicine because of its severe side effects.
o Some human vaccines can be rendered more
effective by precipitating the antigen together with
an aluminum hydroxide salt; e.g. diphtheria toxoid is
used in this form. 8/13/2022
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45. Adjuvant….
47
Synthetic adjuvants usable in humans are currently
being developed.
In some cases, one can take advantage of the Natural
Adjuvant properties of certain vaccines, notably
pertussis;
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46. Adjuvant….
o Triple vaccine, (DPT), consists of alum precipitated
diphtheria toxoid, killed pertussis organisms and
tetanus toxoid.
In this case, the pertussis organisms act as an
adjuvant (much as Mycobacterium does in
Freund's), which increases the immune response to
the two purified toxoid. 8/13/2022
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47. Vaccine Delivery Systems
o Most vaccinations are introduced through the skin:
By scarification (e.g. smallpox) or
By injection (e.g. Salk polio vaccine and many
others)
o The Sabin polio vaccine are administered orally, and
they set up a chronic infection in the gut, stimulating a
local IgA response.
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48. Vaccine Delivery….
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One form of the influenza vaccine (Flumist) is
administered as an intranasal mist, mimicking the
normal route of entry of the infectious organism.
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49. The Ideal Vaccine
o 100% efficient in all individuals of any age
o Provides life-long protection after single
administration
o Does not evoke adverse reactions or cause diseases
o Physically stable under various conditions (temp.,
light, transportation)
o Easy to administer
o Available in unlimited quantities and affordable.
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52. Vaccine Shipments
Receiving Vaccine Shipments
o Arrange for vaccine deliveries to be made only when the vaccine
coordinator or alternate coordinator is on duty (consider holidays,
vacations, staff schedules, and changes in hours of operation).
o All staff members (including non-medical staff) must be aware of
the importance of maintaining the vaccine cold chain and need to
immediately notify the vaccine coordinator of the arrival of
vaccines;
o so that they can be handled and stored properly.
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53. Vaccine Shipments……
o Examine the shipping container and its contents for
any signs of physical damage.
o Cross check the contents with the packing slip to be
sure they match.
o Check the vaccine lot No. and expiration dates to be
sure that you have not received any vaccines.
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54. Vaccine Shipments……
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o Check that the correct amount and type of diluents
have been shipped.
o Check the hot/cold temperature strips.
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55. Vaccine Shipments……
o Vaccines that require diluents will arrive in the
same shipping container.
o For varicella-containing vaccines, the diluents
should be in a separate compartment of the same
container.
o Check that the vaccines were stacked properly.
o Immediately store vaccines in the proper vaccine
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56. Vaccine Shipments……
o If there are any discrepancies with the packaging
slip or concerns about the shipment:
Identify the vaccines with a marking or label,
Separate from other vaccines,
Store the vaccines under appropriate conditions,
Do not use, and
Call the immunization program for guidance.
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57. Vaccine Storage and Handling Guidelines
o Develop and maintain detailed written protocol for staff to
follow
o Assign primary vaccine management responsibilities to one
person
o Provide staff training on vaccine storage and handling.
o All staff assigned to vaccine storage and handling
responsibilities must complete a vaccine storage and
handling training. 8/13/2022
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58. Vaccine Storage Requirements
o Maintain required temperature range at all times
o Stand alone refrigerator units are recommended for
refrigerated vaccines
o Stand alone freezer units are recommended for frozen
vaccines
o Vaccine storage unit must be large enough to hold year’s
largest vaccine inventory.
o Vaccine storage units must be dedicated to vaccines only.
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59. Vaccine Storage Requirements…
o Water bottles can also be stored in the doors to help
stabilize refrigerator temperatures.
o NEVER store vaccines in the door or other compartments
in the refrigerator/freezer.
o Best storage practice–place vaccines in center of fridge
space, 2-3 inches from refrigerator/freezer walls.
Avoid storage on top shelf-near cooling vent or on the
bottom shelf. 8/13/2022
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60. Stock vaccines using
shorter expiration
dates first
Store only vaccines
in the refrigerator
Never leave
vaccine outside the
refrigerator
Don’t store vaccines
on door shelves, in
vegetable/fruit bins
or drawers
Check and log
temperature twice
a day
Store full water
bottles of water on
the bottom shelves
and doors to help
maintain temperature
Open the door
only when
necessary
Keep refrigerated
vaccine between 35°F
and 46°F and the
freezer between -58°F
and +5°F
7
Storage of Vaccine Products
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61. Effect of Temperature on Vaccines
o Live vaccines tolerate freezing.
o Live vaccine potency deteriorate rapidly after
removal from freezer and exposure to light.
o Inactivated vaccines are damaged by exposure to
freezing temperatures.
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62. Temperature Monitoring….
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Freezer:- A place in which the temperature is maintained
thermostatically between –20oc and –10oc (- 4oF and 14oF)
Cold:- A refrigerator is a cold place in which the temperature is
maintained thermostatically between 2oc & 8oc (36oF and 46oF).
Cool:- Any temperature between 8oc and 15oc (46oF and 59oF). An
article for which storage in a cool place is directed may, alternatively,
be stored in a refrigerator, unless otherwise specified by the
individual monograph.
Room Temperature:- the To prevailing in a working area (15-30oc).
BY: Tsegaab Y.
63. Temperature Monitoring….
o Take immediate action when the temperature is outside the
recommended range
Contact each vaccine manufacturer
Report when temperatures were last taken and current
temperatures
Keep a log and follow recommendations
Report issue and actions to health vaccine coordinator.
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64. Vaccine Inventory Control
o Conduct a vaccine inventory at least once per month
(weekly inventory is ideal)
o Avoid stocking excessive vaccine supplies.
Limit inventory to a min. 30-day / max. 120-day
supply
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65. Monitoring Expiration Dates
• Rotate stock using shorter expiration dates first
• If vaccines are 4 months from expiring and will not be used
before the expiration date:
Locate another enrolled provider that is willing to have
vaccines transferred for use
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66. Monitoring Expiration……
69
Record transfer information
Use vaccine transport protocols when transferring
vaccines
Opened multi-dose vials cannot be transferred to another
practice.
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68. OUTLINE
Introduction to antibodies: Classes of antibodies
Basic Antibody Structure
Antibody Targets
Polyclonal vs. Monoclonal antibodies (MAbs)
Hybridoma technology and Engineering of Mabs
Nomenclature for Mabs
Application of Mabs and Approved Biological (Mabs).
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69. Introduction
Antibodies are also called immunoglobulins
(Igs):
Constitute the gamma globulin portion of
blood proteins.
Are soluble proteins secreted by activated B
cells
and plasma cells in response to an antigen.
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70. Introduction….
73
Are capable of binding specifically with a
specific antigen.
There are five classes of antibodies: IgD, IgM, IgG,
IgA, and IgE.
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71. Classes of Antibodies
IgD:– monomer attached to the surface of B cells, important
in B cell activation;
IgM:– pentamer released by plasma cells during the
primary immune response;
IgG:– monomer that is the most abundant and diverse
antibody in primary and secondary response;
• crosses the placenta and confers passive Immunity
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72. Classes of Antibodies…..
75
IgA:– dimer that helps prevent attachment of
pathogens to epithelial cell surfaces;
IgE:– monomer that binds to mast cells and
basophils, causing histamine release when
activated.
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73. Basic Antibody Structure
o The basic antibody is a
dimer of dimer (2
heavy chain-light chain
pairs) composed of
repeats of a single
structural unit known as
the “immunoglobulin
domain.”
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74. Basic antibody structure…
Consists of four looping polypeptide chains linked together
with disulfide bonds;
Two identical heavy (H) chains and two identical light (L)
chains;
The four chains bound together form an antibody monomer;
Each chain has a variable (V) region at one end and a
constant (C) region at the other.
Variable regions of the heavy and light chains combine to
form the antigen-binding site.
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75. Antibody Targets
Antibodies themselves do not destroy antigen; they
inactivate and tag it for destruction.
All antibodies form an antigen-antibody (immune)
complex.
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76. Antibody Targets….
79
Defensive mechanisms used by antibodies are:
Neutralization,
Agglutination,
Precipitation, and
Complement fixation.
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77. Polyclonal vs. Monoclonal antibodies (MAbs)
Polyclonal antibodies: antibody preparations from
immunized animals.
Consist of complex mixtures of different
antibodies produced by many different B cell
clones.
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78. Polyclonal vs. Monoclonal antibodies…….
81
Monoclonal Antibody: homogeneous antibody
preparations produced in the laboratory.
Consist of a single type of antigen binding site,
produced by a single B cell clone.
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79. Polyclonal vs. Monoclonal antibodies…..
82
Mabs are complex proteins that have a uniform basic
structure, comprising four subunits that are divided
into two matched pairs of protein material—two
heavy chains and two light chains, linked by disulfide
bridges, forming a ‘‘Y’’ configuration.
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80. Hybridoma technology, Mouse MAbs,
Historically, Mabs have been produced in
biotechnology in a mouse, and are targeted against
human antigens.
The mouse will create highly specific Mabs against
the human antigen (target); however, the quantity of
Mabs (protein) produced by even very large numbers
of mice is very small;
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81. Hybridoma technology, Mouse MAbs,…..
85
Hence, biotechnology for Mabs also uses a
myeloma cell and fuses it with the mouse
plasma cell to create a murine hybridoma
cell.
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BY: Tsegaab Y.
82. Hybridoma technology, Mouse MAbs,…..
Myeloma cells impart several additional
characteristics to the hybridoma;
o very high production of proteins (Mabs); and
o Long lifespan.
This make Mab manufacturing commercially
feasible;
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83. Hybridoma technology, Mouse MAbs,…..
87
These hydridoma cells are the new master working
cells to produce large amounts of specifically
targeted Mabs.
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84. Hybridoma technology, Murine Mabs
The murine origin of mabs creates significant limits
for these products, because administration of murine
mabs to humans leads to the body’s rejection against
the murine nature of the protein, an immune reaction.
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85. Hybridoma technology, Murine Mabs….
89
Two limits of murine mabs are:
A toxic human antibody mouse antigen (hama)
response with fever and chills; and
Less binding of the mabs to the target cell, thus
limiting their activity.
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86. Hybridoma technology, Murine Mabs….
o Immunogenicity is a complex subject with Mabs, and a
human can react with various types of Mabs against the
Mab being administered, which will neutralize, clear,
sustain, cause HAMA, or cause allergic reactions.
o A patient’s genetics and current immune status will
impact immunogenicity as well as the protein and its
formulation.
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87. Hybridoma technology, Murine Mabs….
91
Mabs is now manipulated by substituting human
subunits for the four murine subunits, creating
chimeric molecules, part murine (about 25%), and
part human (about 75%).
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88. Hybridoma technology, Murine Mabs….
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Even more humanization can be achieved to about 90%,
called ‘‘humanized’’ Mabs, with only the CDRs
(identifying and binding to antigens) being murine.
Furthermore, mice have been bred by genetic engineering
to produce fully human antibodies.
This humanization lessens toxicity and can result in an
increased activity.
BY: Tsegaab Y.
89. Hybridoma technology, Murine Mabs….
Because of the more human Mabs, we now have
been able to expand the number (18 Mab products)
and uses of Mabs to manage inflammatory
conditions, treat cancer, or bind to proteins to arrest
a process, as in slowing kidney rejection in
transplant patients.
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90. Hybridoma technology, Murine Mabs….
94
o Yet another more recent development in antibody
discovery is the use of phage displays instead of
hybridoma technology to create the Mabs.
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91. Engineering of Mabs
o Engineering of Mabs to serve as carriers or fusion
proteins involves consideration of the target antigen, the
antibody-delivery vehicle, and the linker technology.
o The target antigen (Ag) often is a cell surface protein
serving as a cell receptor for cell activation, also called
‘‘CDR’’ complement determining region; any one cell can
have hundreds of CDs on its surface.
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92. Engineering of Mabs…
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The Ag for a Mab should be specific to the desired
antibody(Ab):
With high-binding affinity and expressed in the tumor
Homogeneous at the cell surface,
Expressed throughout the disease process, and not or
minimally expressed in normal tissue.
The antibody should possess accessible sites for loading
the second chemical or product and be able to release
the ‘‘conjugate’’ at the target tissue site.
BY: Tsegaab Y.
93. Engineering of Mabs…
The conjugate being added should not alter:
Ag–Ab binding,
Mab internalization into target cells,
Effector function of the mab such as antibody-dependent cell
cytotoxicity,
Pharmacokinetics of the mab,
Biodistribution of the mab conjugate, or
The aggregation, immunogenicity/ toxicity of the Mab
conjugate.
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94. Engineering of Mabs…
The linkers for the mab and conjugate should
have several desirable properties as well,
such as:
Easy site-specific attachment,
No toxicity, and stability in plasma,
Cleaved intracellularly, and
No immunogenicity.
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95. Engineering of Mabs…
Mabs also are engineered as antibody fragments to be
used alone or combined with other protein as fusion
proteins for their combined actions.
Abatacept (Orencia) combines the fragment of IgG1
and the T-cell antigen protein which creates a protein
molecule with specificity for T-lymphocytes to turn off
the autoimmune reaction in rheumatoid arthritis.
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96. Nomenclature for Mabs
‘tras’ is the name fragment unique
to the Mab protein;
‘‘tu’’ is identified for cancer
indications, vs.
‘‘li’’ for inflammatory conditions,
and
‘‘ci’’ for a cardiovascular indications
or mechanisms of action;
‘‘zu’’ identifies the
‘‘humanized’’ type of Mab
protein vs
mo’’ for fully murine proteins,
‘‘xi’’ for chimeric proteins (75%
human and 25% murine), and
‘‘mu’’ for fully human Mabs;
‘mab’ of course is for monoclonal
antibody.
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97. Application of MAbs
Mab indicated for:
Treatment of rejection of organ transplants,
Prevention of blood clots,
Severe cancers: e.g. Metastatic breast cancer, non-hodgkins
lymphoma, leukemias, and
Inflammatory disease of the gastrointestinal system and
rheumatology areas: e.g., Crohn’s disease and rheumatoid
arthritis,
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98. Application of Mabs…
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Neurologic disease: e.g., Multiple sclerosis, and viral
pneumonia in children.
In oncology, Mabs also serve as carriers of
radioactive species to enhance cell kill;
e.g., tositumomab I-131
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99. Application of Mabs…
The substantial growth in Mab products with major new
indications is predicated on the specificity of Mabs to their cell
targets and especially on the process of humanization of the
murine antibodies, leading to less side effects and more affinity
for the target receptors (more potential desired activity).
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100. Application of Mabs…
o Mabs are also developed for diagnostic testing:
OncoScinct for colorectal and ovarian cancer,
carcino-embryonic antigen (CEA) scan for colorectal cancer,
ProstaScinct for prostate cancer,
MyoScinct for myocardial infarction,
Tecnamab Kl for melanoma,
Verluma for lung cancer,
LeukoScan for osteomyelitis, and 8/13/2022
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