Monoclonal Antibodies As Drug Delivery System

By
Dr. Sunit Kumar Sahoo
 ANTIBODIES

 Antigens (or immunogens) are defined as substances that induce an immune

response.
 The immune response produced may be an antibody (humoral) or production of
sensitized cells (cellular response).
 Proteins formed in response to an immunogen are defined as antibodies.(Constitute
the gamma globulin portion of blood proteins)
 Antibodies are produced by B lymphocytes as one component of the immune
response following recognition of foreign substances (antigens), such as bacterial or
viral proteins.
 When an foreign antigen enters the body due immune response B-Lymphocytes
develops into plasma cells and liberates antibodies or immunoglobulin's of various
types(Ig A, Ig D, Ig E, Ig G, Ig M).
 Classes of antibodies:
There are five classes of antibodies: IgD, IgM, IgG, IgA, and IgE
1. IgD–monomer attached to the surface of B cells, important in B cell
activation
2. IgM–pentamer released by plasma cells during the primary immune
response
3. IgG–monomer that is the most abundant and diverse antibody in
primary and secondary response. crosses the placenta and confers
passive immunity
4. IgA–dimer that helps prevent attachment of pathogens to epithelial cell
surfaces
5. IgE–monomer that binds to mast cells and basophils, causing histamine
release when activated .
•IgG
•Serum concentration: 10 to 16 mg/mL
•Percent of total immunoglobulin: 75%
•Glycosylation (by weight): 3%
•Distribution: intra- and extravascular
•Function: secondary response
•IgG, a monomer, is the predominant Ig class present in human serum

Properties of IgM:
Serum IgM exists as a pentamer in mammals
•Serum concentration: 0.5 to 2 mg/mL
•Percent of total immunoglobulin: 10%
•Glycosylation (by weight): 12%
•Distribution: mostly intravascular
•Function: primary response

•Serum concentration: 1 to 4 mg/mL

•Percent of total immunoglobulin: 15%
•Glycosylation (by weight): 10%
•Distribution: intravascular and secretions
•Function: protect mucus membranes
•IgA exists in serum in both monomeric and dimeric forms,
Properties of IgD:
•Serum concentration: 0 to 0.4 mg/mL
•Percent of total immunoglobulin: 0.2%
•Glycosylation (by weight): 13%
•Distribution: lymphocyte surface
•Function: unknown

Properties of IgE:
•Serum concentration: 10 to 400 ng/mL
•Percent of total immunoglobulin: 0.002%
•Glycosylation (by weight): 12%
•Distribution: basophils and mast cells in saliva and
nasal secretions
•Function: protect against parasites
Structure of antibodies
 Antibodies are glycoproteins comprising 82–96% polypeptides and 4–18%
carbohydrates.
 Proteins with antibody activity are generally called ‘immunoglobulins’.
 Consists of four looping polypeptide chains linked together with disulfide
bonds
a. Two identical heavy (H) chains
b. and two identical light (L) chains (H2L2)
 The four chains bound together form an antibody monomer (Y-shaped)
 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
 Antibodies responding to different antigens have different V regions…

 but the C region is the same for all antibodies in a given class

 C regions (effector regions) form the stem of the Y-shaped antibody and:

 Determine the class of the antibody

 Serve common functions in all antibodies

 Dictate the cells and chemicals that the antibody can bind to

 Determine how the antibody class will function in elimination of antigens

 The bottom trunk portion of antibody molecule is known as Fc region due
to amino acid sequence and is often similar in a given animal species

 The upper arm region : the Fab region , known as variable regions due to its
amino acid sequence which is determined by the antigen responsible for its
formation.

 The variable regions have several hypervariable regions also known as the
Complementary Determining Regions (CDR)
Fab-mediated functions:
1. Antigen recognition – One of the major functions of the Fab region is antigen
recognition.
 The immune system generates large number of antibodies that can
recognize virtually all possible antigens present in pathogens and their
products.
 These may be on invading microbes such as bacteria, viruses, and
parasites as well as environmental antigens.
 Antibodies can be produced against all types of molecules including
carbohydrates, nucleic acids and phospholipids but are best suited to bind
against a protein.
2. Neutralization of pathogens –
 The binding prevents the access of the pathogen into the cells and prevents
infection or destruction of host cells.
 Antibodies also block the binding of the bacteria to host cells by binding to
3. Antibodies are the first line of defence
• IgG also helps in opsonization and complement activation.
•IgG diffuses into the tissues and binds to toxins rapidly.
•IgG can thus neutralize foreign antigens and protect epithelial
cells from infectious agents acting as first line of defence.
Fc-mediated effector functions:
1. Activation of effector cells –
 Through their Fc fragments, antibodies can activate accessory elector
cells.
 These include phagocytic cells like macrophages and neutrophils, T cells
like natural killer cells, and eosinophils and mast cells.
 Thus these cells can identify an Fc fragment and eliminate the
pathogens.
•Complement binding –
•Once bound to the antigen there is formation of antigen–antibody complexes.
• This further activates a complex set of reactions called the complement
cascade. Complements are series of plasma proteins that help in release of
chemical mediators from mast cells (mast cell degranulation), phagocytosis
(eating up of bacterial and microbial cells by macrophages) and cell lysis
(breaking down or bursting of the invading cells).
•Opsonization – Those microbes that replicate outside cells are removed by an
interaction of the Fc part with specific receptors on the surface of elector cells.
•The antibodies coat the surface of the pathogen and allow binding of their Fc
domains to Fc receptors present on elector cells.
•The macrophages and neutrophils then engulf the pathogen and internalize
the microbe causing its destruction.
Antigen- antibody binding
Antibody Targets
Antibodies themselves do not destroy antigen; they inactivate and tag it
for destruction
All antibodies form an antigen-antibody (immune) complex

Defensive mechanisms used by antibodies are

1. Ag-Ab complex formation
2. Neutralization
3. agglutination
4. Precipitation
5. complement fixation
Neutralization –
antibodies bind to and block specific sites on viruses and bacteria, thus preventing these
antigens from binding to receptors on tissue cells
Later destroyed by phagocytes
Agglutination –
antibodies bind the same determinant on more than one antigen
Makes antigen-antibody complexes that are crosslinked into large lattices
(agglutination). IgMs are good at this with mismatched blood
Precipitation –
soluble molecules are cross-linked into large insoluble complexes, fall out of solution,
and are phagocytized
Complement fixation:
Main mechanism used against cellular antigens. Antibodies bound to cells change shape
and expose complement binding sites
This triggers complement fixation on the antigenic cell surface resulting in cell lysis
Classifcation of Antibodies
1. Polyclonal Antibodies
 The immune response to an antigen generally involves the activation of
multiple B-cells all of which target multiple epitopes on that antigen.
 As a result a large number of antibodies are produced with different
specificities and epitope affinities these are known as polyclonal antibodies.
 For production purposes these antibodies are generally purified from the
serum of immunised animals were the antigen of interest stimulates the B-
lymphocytes to produce a diverse range of immunoglobulin's specific to that
antigen.
 The aim is to produce high titre, high affinity antibodies.
 Today these polyclonal antibodies are used extensively for research
purposes in many areas of biology, such as immunoprecipitation,
histochemistry, enzyme linked immunosorbent assays (ELISA), diagnosis of
disease, immunoturbidimetric methods, western blots and Biochip
technology.
Epitope, or antigenic determinant, is a small, specific portion of an
antigen recognized by the immune system such as antibodies.
A single antigen usually has several different epitopes.
The region on an antibody which recognizes the epitope is called a
paratope. Antibodies fit precisely and bind to specific epitopes.
2. Monoclonal Antibodies
Monoclonal antibodies
 represent a single B lymphocyte generating antibodies to one specific
epitope.
 B-cells can be isolated easily from the spleen and lymph nodes of
immunised animals; however, these cells have a limited life span, and can
only divide a limited number of times, coined the 'Hayflick limit’.
 This prohibits the culture of B-cells directly. For an antibody to be useful in
research or industry, it must be readily available in large quantities.
 Due to the Hayflick limit, this would not be possible using B-cells cultured in
vitro as they would eventually stop dividing and the population would die out.
 Consequently, in 1975 Kohler and Milstein developed a technology to fuse immortal
heteromyleoma cells with lymphocytes, using poly ethylglycol (PEG) to break down
cell membranes and allow mixing of the genetic material from both cell types. The
resulting cell type is called a hybridoma.

 This hybridoma takes on the characteristics of both the lymphocyte and

heteromyeloma cell, creating an immortal cell with the ability to produce antibody. As
the new cell line hybridoma is a product of the fusion of one heteromyeloma cell with
one B-cell, the culture only ever has one antibody within the supernatant which, when
purified, is called a Monoclonal antibody.

 This technology allows scientists to extract and purify one antibody from the complex
mixture of antibodies present in the in vivopolyclonal response. This cell line, once
stabilised via single cell cloning, can be frozen and stored indefinitely under liquid
nitrogen, allowing the antibody to be produced in vitro, in large quantities when
required.

 Monoclonal antibodies can be raised against many targets. Specific antibody

characteristics can be identified and selected e.g. sensitivity requirements and cross
reactivity levels can be specified and monoclonal antibodies screened to identify any
cell lines exhibiting the required characteristics.
Antibody Fragments
There are a range of applications in which Fc mediated effects are not required
and are even undesirable.
A common solution for applications where the antibody is only being used to
block a signalling molecule or receptor is the use of antibody fragments that
lack the Fc domain
(1). This also helps to reduce the other main failure of therapeutic antibodies,
namely the lack of delivery, which is especially true for anti-cancer antibodies.
Solid tumours have substantial physical barriers often preventing the
penetration of antibodies to the centre and resulting in reduced therapeutic
effects
(2). The use of smaller fragments enables deeper penetration with the affinity of
the antibody also being critical and if it is too high this will restrict its ability to
penetrate a tumour
3). Fragments are also useful in imaging and cancer therapy, where a long

serum half-life mediated by Fc interaction with the FcRn receptor results in poor
contrast and in the case of radiolabelled antibodies fast clearance from the
circulation via the kidney is also advantageous to reduce prolonged exposure

(4). Antibody fragments that have been engineered to be multimeric are of use
when targeting multiple disease associated antigens.
Over the past three decades antibodies have been dissected into smaller antigen
binding fragments, initially by proteolysis and later by genetic engineering to produce
mono or multivalent fragments.
The image above shows a selection of the antibody fragments that have been
engineered.
These are mainly based on either Fab fragments or the single chain Fv (scFv) as
building blocks.
The Fv portion of an IgG, consisting of the VH and VL domains, is the smallest
fragment that maintains the full binding capacity of the intact antibody.
However, Fv domains tend to have limited stability due to the dissociation of
the domains and thus a peptide linker was introduced between the VH and VL
domains to create the scFv (5, 6).
Single-chain Fv fragments (scFvs) are monovalent structures, with affinity for a single
antigen. Approximately 25 kDa in size, an scFv contains the V regions of an antibody’s
heavy and light chains fused into a single polypeptide chain via a short flexible linker.
An scFv comprises the complete antigen-binding site of its parental antibody molecule
[41] but, lacking the Fc region, is unable to initiate effector functions [42] or bind as
appreciably as intact Abs to Protein .
 scFv’s are currently viewed as promising therapeutic fragments, with several having
been in clinical trials [3,7,9,46]. Recently scFv fragments which can be internalized
by cells were suggested as potentially novel agents for tumor modulation
 ScFvs naturally multimerise (7) and this can be controlled by linker length creating
dimeric, trimer or tetrameric forms (8, 9). For this reason scFvs have become one of
the main building blocks of antibody fragments.
drawbacks of most antibody fragments for use as
therapeutics is the lack of an Fc domain.
The lack of an Fc domain is one reason Ab-fragments exhibit
reduced circulation half-lives and immune system activation

This has led to more recent efforts to either retain this natural
property of IgG or reinstate it by further engineering.

 Examples of the former approach include the one-armed

MetMAb developed by Genentech, which contains a
heterodimeric Fc with only one Fab arm (18),
An example of the latter approach includes

 The use of engineered albumin binding domains fused to the antibody

fragment to infer an albumin-like half-life

 “PEGylation” can enhance Ab-fragment circulation life [3,12–15]. Cimzia® is

one example of a PEGylated Ab fragment.

 Polysialylation: Polysialic acid (PSA) is a polymer derived from N-

acetylneuraminic (sialic) acid) and has been used for protein conjugation
[67,68]. The hydrophilic nature of PSA is thought to result in similar
hydration properties to PEG, giving it a high apparent molecular weight in
the blood and therefore increased circulation time.
 Bispecific antibodies (bsAbs) combine specificities of two antibodies and
simultaneously address different antigens or epitopes.
 BsAbs with ‘two-target’ functionality can interfere with multiple surface receptors or
ligands associated

Fig.. The upper two lines depict immunoglobulin (Ig)-like bsAbs comprising an IgG Fc
region, either as bivalent or tetravalent molecules. Furthermore, several small bsAb and
bsAb fusion proteins have entered clinical trials. Abbreviations: BiTE, bispecific T cell
engager; DART, Dual affinity retargeting; DNL, dock-and-lock; DVD-Ig, dual variable
domain immunoglobulins; HSA, human serum albumin; kih, knobs into holes.
 Since bispecific antibodies do not typically occur in nature, they are constructed either
chemically or biologically, using techniques such as cell fusion or recombinant DNA
technologies.
 The ability to bind two different epitopes with a single molecule offers a number of
potential advantages.
 One approach is to use the specificity of one arm as a targeting site for individual
molecules, cellular markers or organisms, such as bacteria and viruses. While the
other arm functions as an effector site for the recruitment of effector cells or delivery
of molecular payloads to the target, such as drugs, cytokines or toxins.
 Alternatively, bispecifics can be used to dual target, allowing detection or binding of a
target cell type with much higher specificity than monospecific antibodies.
 HYBRIDOMA TECHNOLOGY FOR PRODUCTION OF MONOCLONAL
ANTIBODIES

 Hybridomas are cells that have been engineered to produce a desired antibody in
large amounts, to produce monoclonal antibodies.
 Hybridoma technology was discovered in 1975 by two scientists, Georges Kohler of
West Germany and Cesar Milstein of Argentina (now working in U.K.), who jointly
with Niels Jerne of Denmark (now working in Germany) were awarded the 1984
Noble prize for physiology and medicine.
 A hybridoma, which can be considered as a harry cell, is produced by the injection of
a specific antigen into a mouse, procuring the antigen-specific plasma cells (antibody-
producing cell) from the mouse's spleen and the subsequent fusion of this cell with a
cancerous immune cell called a myeloma cell
 The hybrid cell, which is thus produced, can be cloned to produce many identical
daughter clones.
 These daughter clones then secrete the immune cell product.
 Since these antibodies come from only one type of cell (the hybridoma cell) they
are called monoclonal antibodies.
The advantage of this process is that it can combine the qualities of the two different
types of cells;
a. the ability to grow continually,
b. and to produce large amounts of pure antibody.
 
 Immunization
 Cell fusion
 Selection of hybridomas
 Screening the products
 Cloning and propagation
 Characterization and storage

36
 Laboratory animals (eg. mice) are first exposed to an antigen to which we
are interested in isolating an antibody against.
 Once splenocytes are isolated from the mammal, the B cells are fused with
immortalized myeloma cells - which lack the HGPRT(hypoxanthine-
guanine phosphoribosyltransferase) gene - using polyethylene glycol’

 Fused cells are incubated in the HAT (Hypoxanthine Aminopetrin Thymidine)

medium. Aminopterin in the myeloma cells die, as they cannot produce nucleotides
by the de novo or salvage medium blocks the pathway that allows for nucleotide
synthesis.
 Hence, unfused D cell die. Unfused B cells die as they have a short life span.
 Only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the
B cells is functional. These cells produce antibodies (a property of B cells) and are
immortal (a property of myeloma cells). 2

 The incubated medium is then diluted into multi well plates to such an extent that
each well contains only 1 cell.
 Then the supernatant in each well can be checked for desired antibody.
 Since the antibodies in a well are produced by the same B cell, they will be directed
towards the same epitope, and are known as monoclonal antibodies
 
 Immunize an animal usually mouse by injecting with an
appropriate antigen along with Freund’s complete or incomplete
adjuvant.
 Adjuvants are non specific potentiators of specific immune
responses.
 Injection of antigens at multiple sites are repeated several times
for increased stimulation of antibodies.
 3 days prior to killing of animal a final dose is given
intravenously.
 Spleen is aseptically removed and disrupted by mechanical or
enzymatic methods to release the cells.
 By density gradient centrifugation lymphocytes are separated
from rest of the cells.

38
 The first therapeutic monoclonal antibody registered by FDA.
 OKT3 – mouse monoclonal anti-CD3 (1986).
 This therapy is quite effective but its repeated use is accompanied with
severe immunological side effects:

HAMA (human anti-mouse-antibodies)

 The constant part of an Ig is conserved, but there are some differences
between human and mouse Igs.
 HAMA can be detected after 8-12 days of treatment, the peak concentration is after
25-30 days.

43
 murine antibodies are immunogenic to humans, the obvious solution for this is to clone a fully
human antibody. But it has many problems like ethical clearance, difficult to culture, impossible to
obtain many of the appropriate antibodies.

 To over come HAMA(human antimouse antibody) response, a chimeric antibody is prepared with
Fc region of human IgG and Fab regions of murine origin by the use of DNA recombinant
technology.

 The Fv region of the chosen monoclonal antibody gene is fused to Fc part of a human Ig gene.

 Approximately 75 of a chimera Ig % is of human origin.

 The specificity of the antibody is similar to that of the original mouse antibody.

 The in vivo half life and effector functions of the Ig are similar to those of the original human
antibody.

 HACA (human anti-chimeric-antibodies) Less immunogeneicity, but sometimes HACA can be

detected.
 V domains

Mouse

Chimeric

Human
C domains

45
 The genes of the CDR regions of a mouse monoclonal antibody are implanted into
the genes of human antibody.
 More than 90% of the antibody is of human origin.
 The specificity of the humanized antibody is similar to that of the original mouse
antibody.
 The in vivo half life and effector functions of the humanized antibody are similar to
those of the original human antibody.
 Humanized antibodies are prepared by recombinant DNA.
 Thus humanized antibodies are 95% homology with human antibodies.
 The first humanised antibody was alemtuzumab (Campath®, anti-CD52),
now used for treatment of leukaemia.
 hypervariable

Mouse

Humanised

Human
framework

47
 
Bispecific antibodies:
 These are specific to two types of antigens.
 They are constructed by r.DNA technology.
 Each arm is specific to one type of antigen.

49
 For MAb targeted drug delivery, a drug is bound covalently to an
antibody that is chosen to target it to the desired site of action.

 Spacer is present between the antibody and the drug.

 Drug is non-covalently incorporated into a liposome or microsphere to

which the targeting antibody is bound to the surface—immunoliposome
or immunomicrosphere resp.

50
Applications of monoclonal antibodies
(1) Diagnostic Applications

(2) Therapeutic Applications

(3) Protein Purification and
(4) Miscellaneous Applications.
1. Diagnostic Applications:
Monoclonal antibodies have revolutionized the laboratory diagnosis of various
diseases. For this purpose, MAbs may be employed as diagnostic reagents for
biochemical analysis or as tools for diagnostic imaging of diseases.
(A) MAbs in Biochemical Analysis:
Diagnostic tests based on the use of MAbs as reagents are routinely used in
radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA) in the
laboratory.
These assays measure the circulating concentrations of hormones (insulin, human
chorionic gonadotropin, growth hormone, progesterone, thyroxine,
triiodothyronine, thyroid stimulating hormone, gastrin, renin), and several other
tissue and cell products (blood group antigens, blood clotting factors, interferon’s,
interleukins, histocompatibility antigens, tumor markers).
In recent years, a number of diagnostic kits using MAbs have become
commercially available. For instance, it is now possible to do the early diagnosis
of the following conditions/diseases.
Pregnancy
by detecting the urinary levels of human chorionic gonadotropin.
Cancers:
Cancers estimation of plasma carcinoembryonic antigen in colorectal cancer,
and prostate specific antigen for prostate cancer
Hormonal disorders
analysis of thyroxine, triiodothyronine and thyroid stimulating hormone for
thyroid disorders.
Infectious diseases:
Infectious diseases by detecting the circulatory levels of antigens specific to the
infectious agent e.g., antigens of Neisseria gonorrhoeae and herpes simplex
virus for the diagnosis of sexually transmitted diseases.
B) MAbs in Diagnostic Imaging:
Radiolabeled—MAbs are used in the diagnostic imaging of diseases, and this
technique is referred to as immunoscintigraphy.
The radioisotopes commonly used for labeling MAb are
1. iodine—131
2. technetium—99.
The MAb tagged with radioisotope are injected intravenously into the patients.
These MAbs localize at specific sites (say a tumor) which can be detected by
imaging the radioactivity.
In recent years, single photon emission computed tomography (SPECT) cameras
are used to give a more sensitive three dimensional appearance of the spots
localized by radiolabeled— MAbs.
Immunoscintigraphy is a better diagnostic tool than the other imaging techniques
2. Therapeutic Applications:
Monoclonal antibodies have a wide range of therapeutic applications. MAbs are used in
the treatment of cancer, transplantation of bone marrow and organs, autoimmune
diseases, cardiovascular diseases and infectious diseases.
The therapeutic applications of MAbs are broadly grouped into 2 types:
(A) Direct use of MAbs as therapeutic agents
(B) MAbs as targeting agents.
MAbs as Direct Therapeutic Agents:
Monoclonal antibodies can be directly used for enhancing the immune function of the
host. Direct use of MAbs causes minimal toxicity to the target tissues or the host.

In destroying disease-causing organisms:

MAbs promote efficient opsonization of pathogenic organisms (by coating with
antibody) and enhance phagocytosis. In fact, MAbs were found to protect chimpanzees
against certain viral (hepatitis B-virus) and bacterial (E. coli Haemophilus influenza,
Streptococcus sp and Pseudomonas sp) infections.
In the treatment of cancer:
MAbs, against the antigens on the surface of cancer cells, are useful for the treatment of
cancer. The antibodies bind to the cancer cells and destroy them.
This is brought out by antibody—dependent cell-mediated cytotoxicity, complement-
mediated cytotoxicity and phagocytosis of cancer cells (coated with MAbs) by
reticuloendothelial system.
The patients suffering from leukemia, colorectal cancer, lymphoma and melanoma have
been treated with Mabs
Over 20 antibody-drug conjugates in clinical trials as well as a recently
FDAapproved
drugs
• Why they are successful?
• Used for selective delivery of highly cytotoxic agents to tumor cells while
sparing normal tissue.
Why linker is important?
What are the important requirements?
• The exact connection between the cytotoxic agent and the antibody has
profound effects on the selectivity, pharmacokinetics, therapeutic index, and
overall success of the ADC.
• The linker must be stable in systemic circulation to allow delivery of the intact
ADC to the targeted antigen presented on the surface of the tumor cell.
• The linker must then be labile enough to allow efficient release of the cytotoxic
drug inside the targeted tumor cell.
In the treatment of AIDS:
MAbs as Targeting Agents in Therapy:
Toxins, drugs, radioisotopes etc., can be attached or conjugated to the tissue-specific
monoclonal antibodies and carried to target tissues for efficient action.
This allows higher concentration of drugs to reach the desired site with minimal
toxicity. In this way, MAbs are used for the appropriate delivery of drugs or isotopes
Immunoconjugate modules. Immunoconjugates are composed of three different
components: a monoclonal antibody, a therapeutic entity, and a linker that joins the
antibody and the therapeutic agent. Using this basic immunoconjugate model, different
therapeutic entities, including pharmacologics, radioactive agents, and toxins, can be
delivered to the cancer cells.
The toxins can be coupled with MAbs to form immunotoxins and used in therapy e.g.,
diphtheria toxin, Pseudomonas exotoxin, toxins used for cancer treatment

 Ricin is a cytotoxic protein derived from castor oil plant. It is composed of two
polypeptide chains (A and B) held together by a disulfide linkage. The B-chain of
ricin binds to the cell surface. This binding facilitates the A-chain of ricin to enter
the cell and inhibit the function of ribosomes (i.e. biosynthesis of all proteins is
blocked).

 This results in the death of cells (Fig. 17.8A). Ricin can be subjected to oxidation to
separate to A and B chains. The toxic A-chain can be conjugated to MAb that is
specific to cancer cells. The tumor- specific MAb bound to A-chain of ricin binds to
cancer cells and not to normal cells. Once the A-chain enters the cells, it blocks
ribosomal function, leading to the death of cancer cells (Fig. 17.8B).
MAbs in the dissolution of blood clots:
A great majority of natural deaths are due to a blockage in cornary or cerebral
artery by a blood clot (thrombus).
Fibrin is the major constituent of blood clot which gets dissolved by plasmin.
Plasmin in turn is formed by the activation of plasminogen by plasminogen
activator.
The blockage of arteries occurs due to inadequate dissolution of blood clots.
Tissue plasminogen activator (tPA) can be used as a therapeutic agent to
remove the blood clots.
MAbs in drug delivery:
In general, the drugs are less effective in vivo (in the living body) when compared to in
vitro (in laboratory when tested with cultured cells).

This is mainly due to the fact that sufficient quantity of the drug does not reach the
target tissue.

This problem can be -solved by using tissue-specific MAbs.

The drugs can be coupled with MAb (directed against a cell surface antigen of the cells,
say a tumor) and specifically targeted to reach the site of action

Agents like chlorambucil, methotrexate and doxorubicin are conjugated with tumor
specific antibodies.

Ex: doxorubicin-BR96 immunoconjugate for Lewis antigen found on the surface of

tumor cells.
3. Protein Purification:
4. Miscellaneous
: Applications:
Catalytic MAbs (ABZYMES)
 Autoantibody Fingerprinting
 
 They are homogenous in nature.
 They are specific to a particular antigen with a
particular epitope.
Ex:Rituximab (Rituxan®, anti-CD20) is a good
example – this antibody is used for the treatment of
lymphoma.

67
 FDA Approval

The first approved mAbs was OKT-3 [1986], which is a
murine IgGa2 protein to deplete T cells in patients with
acute rejection of renal allotransplant.