Immunological Products

II. Antibodies as therapeutic agents

Kinefergb Derash. (B.pharm, M.sc, in

Clinical pharmacy)
Lecturer
Wollo university
 Immunological Products
II. Antibodies as therapeutic agents

• Few substances have had a greater positive

impact upon human healthcare management
 antibodies,
 vaccines and
 adjuvants

• For most of this century, these immunological

agents have enjoyed widespread medical
application,
• predominantly for the treatment/prevention of
infectious diseases.
Immunity –all the mechanisms used by the body as protection
against environmental agents that are foreign to the body.

Type of immunity
• Innate:- derives from all those elements with which an individual is
born and that are always present and available at very short notice
to protect the individual from challenges by ‘foreign’ material.
Physical barriers –skin, mucous membranes
Chemicals- pH, lipids, enzymes lysozymes
Cellular- monocytes, macrophages, Eosinophiles
Acquired immunity :
• More specialized than innate immunity

• The initial contact with the foreign agent (immunization)

triggers a chain of events that leads to
• the activation of certain cells (lymphocytes) and
• the synthesis of proteins (antibodies) with specificity
against the foreign agent.
Acquired immunity
• Cells involved in acquired immunity
Humoral Cells
• B cells ---------Antibodies
Cellular component
• T cells---------lymphokines
Macrophages
• Involved in the presentation of the foreign material to T cell.

 Unlike innate immunity, acquired immunity exhibits specificity :

 it develops only against that substance which was used for
immunization.
Humoral immunity
• Is mediated by serum antibody.

• Antibodies are proteins that bind very tightly to their targets

(antigens). They are produced as a defense against infection.

• Antibodies are a heterogeneous mixture of serum globulins, all of

which share the ability to bind individually to specific antigens.

• All serum globulins with antibody activity are referred to as

immunoglobulins (Ig).
Antibody structure
All immunoglobulin molecules have many common structural
features, but they differ from one another in that portion of the
molecule that binds specifically to the respective antigen.

• The greatest variability of

immunoglobulins occurs at the N-
terminal, or the tips, of the “Y”
molecule.

• This variable region includes the

ends of both the heavy (VH) and
the light (VL) chains.
• In addition to differences in the antigen-binding portion of
different immunoglobulin molecules,
• there are other differences, the most important of which are
those in the H chains.
 constant regions
their immune function

IgG, IgM, IgA, IgE, and IgD

• IgG is the most abundant class of immunoglobulins in sera.

 antigens - multiple epitopes,

• a variety of B-cell clones are induced

• polyclonal antibody - heterogeneous mixture of antibodies,

each specific to one epitope.

• The antibodies produced from a single B-cell clone are

monoclonal in that they are all specific to a single epitope
 Antibody specificity
• An individual animal can make
billions of different antibody
molecules, each with a distinct
antigen-binding site. Each
antibody recognizes its antigen
with great specificity.

For any antigen that enters, the B

cells can produce antibodies with
the exact complementary shape and
size to fit them.
 Use of Antibodies

• Antibodies have been used as research tools for

cell, antigen, or pathogen identification;

pathogenesis studies;
ligands for column chromatography and molecule purification;
diagnostic reagents;
therapeutic antibody preparations; and
the identification of protective antigens/epitopes in vaccine
development.
 Antibodies as therapeutic agents

POLYCLONAL ANTIBODIES

Preparation
• Most antigens offer multiple epitopes and therefore induce
proliferation and differentiation of a variety of B-cell clones,
each derived from a B-cell that recognizes a particular epitope.

• The resulting serum antibodies are heterogenous (polyclonal

antibodies), comprising a mixture of antibodies, each specific for
one epitope.
Therapeutic use of Polyclonal antibodies
• Polyclonal antibody preparations have been used for several
decades to induce passive immunization against infectious
diseases and other harmful agents, particularly toxins.

• Direct IV injection

• Immediate immunological protection but Transitory

effect (persist 2 – 3 weeks)
Passive immunization

• Prophylactically (i.e. to prevent a future medical episode)

 prior administration of a specific anti-snake toxin antibody

preparation to an individual before they travel to a world
region in which these snakes are commonly found.

• Therapeutically (i.e. to treat a medical condition that is already

established)
administration of the anti-venom antibody immediately
after the individual has experienced a snake bite.
Sources of polyclonal antibodies

• Antibody preparations used to induce passive immunity may be

obtained from either
 animal
antisera

 human sources
immunoglobulin preparations

In both cases, the predominant antibody type present is IgG.

Production of polyclonal antibodies

• Antisera
immunizing healthy animals (e.g. horses) with appropriate
antigen.

 Regular withdrawal of small samples of blood

 quantitatively analysed
 facilitates harvesting of the blood at the most
appropriate time points.
• repeated antigen booster injections.
Collection of the PCA
• Aseptically into sterile containers
• There are two techniques
1. Allowing to clot
• the antibody-containing container - centrifugation.
 Antisera
2. The blood may be collected in the presence of heparin
• Removal of the suspended cellular elements, again by
centrifugation.
 the resultant antibody-containing solution is
termed ‘plasma’.
Traditional Polyclonal Antibody Preparations

Figure: Overview of the production of antisera for therapeutic use to

induce passive immunization.
Problem (of using animals as PCA sources)

induce unwanted side effects.

their ability to induce hypersensitivity reactions;

 serum sickness -not acute
 anaphylaxis -life threatening

• Because of such risks, antibody preparations derived from

human donors (i.e. immunoglobulins) are usually preferred as
passive immunizing agents.
• Immunoglobulins are purified from the serum (or plasma) of
human donors by methods similar to those used to purify animal-
derived antibodies.

• In most instances, the immunoglobulin preparations are enriched

in antibodies capable of binding to a specific antigen

• These may be purified from donated blood of individuals who

have recently:

 been immunized against the antigen of interest;

 recovered from an infection caused by the antigen of
interest.
• The major polyclonal antibody preparations used
therapeutically are listed in the next slide.
• These may generally be categorized into one of several
groups upon the basis of their target specificities. These
groups include antibodies raised against:

specific microbial or viral pathogens

microbial toxins

snake/spider venoms (anti-venins).

Table: Polyclonal antibody preparations of human or animal origin
used to induce passive immunity against specific biological agents
Polyclonal antibody

• Advantages - the antibody heterogeneity increases immune

protection in vivo
the localization
phagocytosis and
complement-mediated lysis of antigen.

• Disadvantage - often reduces the efficacy of an antiserum for

various in vitro use.

NB: for most research, diagnostic, and therapeutic purposes,

monoclonal antibodies, derived from a single clone and thus
specific for a single epitope, are preferable.
 Monoclonal Antibodies

Monoclonal antibodies: identical antibodies that are all descendants

of one specific B cell. One type of antibody is produced for each type
of antigen
Production of monoclonal antibodies

• For decades, scientists investigated ways of manufacturing

antibodies by culturing white blood cells outside the body.
• But the white blood cells wouldn’t divide outside the
body.

• The breakthrough came in 1975 when scientists devised a

technique to produce clones of a single type of antibody,
known as monoclonal antibodies.

• In the last 20 years or so, antibody-based therapeutics have

mainly focused upon the medical application of monoclonal
antibodies.
 Monoclonal antibody technology (Hybridoma technology )
-Kohler and Milstein (1975)
• Inputs

Myeloma cells antibody-producing cells

B-lymphocytes ( ‘hybridoma’ cells)
Propylen glycol (stable, cancerous)

Inexhaustible source of mono-specific (monoclonal) antibody.

• Hybridoma technology facilitates the relatively straightforward

production of mono-specific antibodies against virtually any
desired antigen.
 Application of Monoclonal Antibodies

• The unrivalled specificity of monoclonal antibodies, coupled to

their relatively straightforward production and their continuity of
supply, renders them attractive biochemical tools.

• Therapeutically, they represent by far the single largest category

of biopharmaceutical substances under investigation.

• Several hundred such preparations are currently undergoing

preclinical and clinical trials
• Throughout the 1980s the focus of attention rested upon
their use either as in vivo imaging (i.e. diagnostic) agents
or as direct therapeutic agents.

• Initial studies centered mainly around cancer, but

monoclonal antibody preparations are now used in a
variety of other medical circumstances:

induction of passive immunity;

diagnostic
therapeutically (e.g. treatment of cancer).
• All
• in vivo diagnostic
• therapeutic
• drug delivery applications
• are dependent upon the selective interaction of a monoclonal
antibody with a specific target cell type in the body (e.g. a cancer
cell).

• Therefore, a prerequisite to application of monoclonal antibody-

based products in this way is the identification of a cell surface
antigen unique to the target cell type.
Figure Underlying principle/approaches taken during the development and
use of antibody-mediated target cell detection/destruction. A prerequisite
for adoption of this strategy is the identification and characterization of a
surface antigen unique to the target cell type (‘unique surface antigen’, USA)
(a). Antibodies raised against the USA should selectively interact with the
target cell (b). In some instances the antibody is chemically coupled to a
radioactive tag (c), a drug or a toxin (d)
Schematic illustrating an antibody-drug conjugate
 Mouse monoclonal antibodies

• Antibody immunogenicity remains one of the inherent

therapeutic limitations associated with administration of
murine monoclonals to human subjects.

• In most instances, a single injection of the murine monoclonal

will elicit an immune response in 50–80 per cent of patients.

• Human anti-mouse antibodies (HAMA) will generally be

detected within 14 days of antibody administration.
• Repeated administration of the monoclonal (for therapeutic
purposes) will increase the HAMA response significantly

 It will also induce an HAMA response in the majority of

individuals who display no such response after the initial
injection

• The HAMA response will effectively and immediately destroy

subsequent doses of monoclonal administered

• In practice, therefore, therapeutic efficacy of murine

monoclonals is limited to the first and, at most, the second dose
administered
 Proposed solutions HAMA

• It took the scientists at the Medical Research Institute in

Cambridge 11 years to solve this problem.

• rDNA technology has provided an alternative (and successful)

route of reducing the innate immunity of murine monoclonals.

• The genes for all human immunoglobulin sub-types have been

cloned
• has allowed generation of various hybrid antibody
structures of reduced immunogenicity.
1. Humanized monoclonal antibodies
using rDNA
2. Chimaeric

• A better understanding of antibody disposition and technical

developments in antibody engineering have provided essential
knowledge for transforming mouse monoclonal antibodies
into chimeric and humanized forms with improved safety and
efficacy.
 1. Chimaeric

• Chimaeric antibodies consist of murine monoclonal VH and VL

domains grafted onto the Fc region of a human antibody.

• The chimaeric antibody would display the specificity of the

original murine antibody, but would largely be human in
sequence.
Figure: Production
of chimaeric
• It was hoped that such chimaeric antibodies, when compared
with murine antibodies, would be:
 significantly less immunogenic;
 display a prolonged serum half-life;

Table: The serum half-life values of some IgG antibody preparations when
administered to humans

 allow activation of various Fc-mediated functions.

2. Humanized monoclonal antibodies

• consists of murine (complementarity-determining region )CDR

regions grafted into a human antibody

• These antibodies are 90% human and don’t provoke an immune

response but are still capable of binding to the antigen.
Figure: Production of
humanized antibodies
Differences Between Small Molecules and Therapeutic
Monoclonal Antibodies that Can Affect Clinical Development
 Diagnosis application

Monoclonal antibodies
• particularly useful in diagnostic areas where specificity is the
most important criterion,
for example,
• in the measurement of circulating steroid hormones
• in discriminating between viral strains.
• analysis of disease phases, such as those associated with
developmental stages of parasite life cycles characterized
by alteration in the expression of cell surface antigens.
• for the diagnosis of pregnancy using rapid, simple, and
direct assays
 Therapeutic application

• The potential for MABs as therapeutic agents was quickly

recognized and the first MAB was approved for therapeutic
use in 1986
• MABs can be used directly to induce an inflammatory
response to tumor cells, or to block receptors;
• they can be conjugated with a cytotoxic or radioactive
molecule such as Iodine 131
• By attaching liposomes containing cytotoxic drugs to single
chain antibody constructs, anticancer drugs can be delivered
effectively to the cell interior.
• The antibody can act in several ways including the induction
of apoptosis, the blocking of growth factor receptors,
induction of cell- or complement-mediated cytotoxicity, or the
blocking of angiogenesis.
• However, direct actions require the antibody to penetrate the
tumor mass.
• This may be facilitated by the use of antibody fragments,

• but the combination of immunotherapy with surgery and

radio- or chemotherapy has been demonstrated to be the
most effective approach
• the prevention of viral or bacterial infection by direct
immuno-neutralization (the blockading of viral entry by
preventing binding to receptors) or the stimulation of
endogenous antibody responses
• to remove a specific substance from the circulation, including
• the acute elimination of venoms or toxins following, for
example, snakebite.
• Accidental or deliberate overdoses of drugs or poisons,
such as paraquat, can also be treated.
 APPLICATIONS OF MONOCLONAL ANTIBODIES IN DRUG
DELIVERY

Principle of Targeting
• Several classes of drugs lack specificity for diseased cells;
for example, the cytotoxic action of chemotherapeutic agents
• cause serious side effects.
• One way of circumventing this problem is to deliver the drug
in a manner such that it is preferentially localized at the
desired site of action, or it predominantly attacks the diseased
cells.
• This process is called targeting.
• Targeted drug delivery systems can be classified into three
categories; passive, physical, or active targeting.
• Passive targeting refers to the natural in vivo distribution
pattern of the drug delivery system, which is determined by the
inherent properties of the carrier
• physical targeting, some characteristics of the environment are
utilized to guide the carrier to a specific site or to trigger
selective release of its content at the site.
• Usually, it is accomplished via an external mechanism, such as
• induced local hyperthermia (e.g., using thermally sensitive
liposomes) or
• a localized magnetic field (e.g., using magnetically
responsive albumin microspheres).
• active targeting, the natural disposition pattern of a carrier is
modified to target it to specific organs, tissues, or cells.
• cell-specific ligands have been used
• MABs are applicable mode of active targeting.
 Drug delivery

• The drug can be covalently bound to the MAb directly or the

two can be conjugated through a linker such as a water-
soluble polymer.
• Alternatively, a carrier such as a liposome or a polymeric
microsphere can be used, wherein the drug is entrapped in or
bound to the carrier, and the MAb is bound to the surface of
the carrier.
• upon reaching the target, either the immunoconjugate itself
should have the desired pharmacological effect equivalent to
the free drug, or must release free drug or a derivative that is
fully efficacious
• Plasma or intracellular enzymes
• local pH of the target tissue or of its intracellular
environment (e.g., lysosomes) may also increase the rate
of release of drug from the immunoconjugate.
THE END!