MONOCLONAL ANTIBODIES OF

CLINICAL SIGNIFICANCE

Dr.Vignesh.SResident in Internal

MedicineGuided by

Prof.Dr.R.L.Meena

1) History and Discovery2) Monoclonal Antibodies3) Production and

Nomenclature4) Advantages and

Disadvantages

5) CLINICAL SIGNIFICANCE1)DIAGNOSTIC2)THERAPEUTIC3)RESEARCH

Plan of the Seminar

6)FDA approved MAbs7)Problems faced with

old generation MAbs8)How was it overcome9)LATEST UPDATE on

MAbs10)Conclusion

ABBREVIATION USED:

MAb Monoclonal Antibody

Humans have the ability to make antibodies able to◦recognize (by binding to) virtually any antigenic

determinant (epitope)◦to discriminate between even similar epitopes

The remarkable specificity of antibodies makes them promising agents for human therapy.

ANTIBODIES IN HUMAN THERAPY

But there are problems to be solved before antibodies can be used in human therapy.

The Polyclonal Response Limitation of the Growth Potential

PROBLEM FACING…

What was needed was a way◦To make antibodies of a single specificity

◦To make antibodies all built alike manufactured by a single clone of cells

◦To make antibodies that can be grown indefinitely.

WHAT NEXT…???

To make An antibody that will bind only to one type/group of cells in a

patient Coupling a cytotoxic agent (e.g. a strong radioactive isotope)

to that antibody, and Then giving the complex to the patient so it can seek out and

destroy the only particular type of cells (and no normal cells).

THE TARGET

THE MYELOMA THEORY In the 1970’s the B-

cell cancer myeloma was discovered, and it was understood that these cancerous B-cells all produce a single type of antibody.

This was used to study the structure of antibodies, but it was not possible to produce identical antibodies specific to a given antigen.

This problem was solved in 1975 with a technique deviced by Köhler , Milstein and Jerene

The technique is called somatic cell hybridization.

The result is a hybridoma cell producing antibodies targeting a single epitope in large numbers

THE DISCOVERY

Nobel Prize Award in Medicine and Physiology in 1984

Prof.Georges J. F. Köhler Prof.César

MilsteinProf.Niels Kaj

Jerne

 They are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell

They have monovalent affinity, in that they bind to the same epitope.

Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect that substance.

MONOCLONAL ANTIBODIES

lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT).◦ This enzyme enables cells to synthesize purines using

an extracellular source of hypoxanthine as a precursor.

◦ Ordinarily, the absence of HGPRT is not a problem for the cell because cells have an alternate (de novo)pathway that they can use to synthesize purines.

◦ However, when cells are exposed to aminopterin (a folic acid analog), they are unable to use de novo pathway and are now fully dependent on HGPRT for survival.

MYELOMA CELL

B cell has the enzyme HGPRT But B cells die soon They do not have the capacity to grow

indefinitely because of their limited life span

B cell

Scanning Electron Microscopic view of a B cell

CELLS FUSED:◦Spleen cells from a mouse that has been

immunized with the desired antigen◦Myeloma cells.

FUSION AGENT:◦Polyethylene glycol

MEDIUM:◦HAT Medium {Hypoxathine-Aminopterin-

Thymidine}

THE PROCEDURE

VIDEO

Courtesy:Dr.Bhanu Prakash

Every monoclonal antibody has the following components in its name

Variable-Target Substem-Source Substem-Stem-Additional words(in special cases)

Ex: Alacizumab pegol is

Ala –ci-zu-mab-pegol

NOMENCLATURE

OLD NEW MEANING

-anibi- - Angiogenesis inhibitor

-ba(c)- -ba- Bacterium

-ci(r)- -ci- Circulatory system

-fung- -fu- Fungus

-ki(n)- -ki- Inlterleukin

-les- - Inflamattory lesions

-li(m)- -li- Immune system

-mul- - Musculoskeletal system

-ne(ur)- -n(e)-* Nervous system

-os- -s(o)- bone

-toxa- -tox(a)- toxin

- -tu- Tumour

-vi(r) -vi- virus

Target Substem

Letter Meaning

-a- Rat

-e- Hamster

-i- Primate

-o- Mouse

-u- Human

-xi- Chimeric

-zu- Humanized

-axo- Rat/mouse hybrid

Source Substem

Rituximab◦Ri- Variable◦tu- Tumour◦xi- Chimeric◦mab-Monoclonal Antibody

So Rituximab is a Chimeric Monoclonal Antibody targetting a Tumour

Example 1

Bevacizumab◦Beva- Variable◦ci- Circulatory System◦zu- Humanised◦mab- Monoclonal Antibody

So Bevacizumab is a Humanised Monoclonal Antibody targetting a protein in Circulatory System

Example 2

A second word following the name of the antibody indicates that another substance is attached.1)An antibody can be PEGylated (attached to molecules of polyethylene glycol)

◦ to slow down its degradation by enzymes and to decrease its immunogenicity

Ex:alacizumab pegol2)A cytotoxic agent can be linked to an anti-tumor antibody for drug targeting purposes.

◦ vedotin,(monomethyl auristatin E) which is toxic by itself but predominantly affects cancer cells if used in conjugates

Ex:glembatumumab vedotin3)A chelator for binding a radioisotope can be attached. 

◦ Pendetide, a derivative of pentetic acid, in capromab pendetide to chelate indium-111.If the drug contains a radioisotope, the name of the isotope precedes the name of the antibody.

Ex: indium (111In) capromab pendetide 

Additional words

1)Homogeneity: Monoclonal antibody represents a single antibody molecule that

binds to antigens with the same affinity and promote the same effectors functions.

2) Specificity: The product of a single hybridoma reacts with the same epitope on antigens.

3) Immunizing Antigen:Need not be characterized and is ultimately not needed in large quantities to produce large quantities of antibody.

4) Selection: It is possible to select for specific epitope specificities and generate antibodies against a wider range of antigenic determinants.

5) Antibody Production:Unlimited quantities of a single well-defined monospecific reagent

ADVANTAGES

Average affinity of monoclonal antibodies are generally lower than polyclonal antibodies

Monoclonals against conformational epitopes on native proteins may lose reactivity with antigens.

Antibodies sometimes display unexpected crossreactions with unrelated antigens.

Immune rejections Time and effort commitment: VERY LARGE.

DISADVANTAGES

CLINICAL SIGNIFICANCE

They are used in  Western blot test ELISA TEST Antigen capture assays Immuno dot blot tests to detect the specific protein on a membrane. Naked eye dipstick tests Immuno-histochemistry , which detect antigen in fixed tissue

sections and Immuno-fluorescence test, which detect the substance in a frozen

tissue section or in live cells. Radio immuno assays Tissue typing Serotyping of Microorganisms They are also used in the diagnosis of lymphoid and myeloid

malignancies

DIAGNOSTIC SIGNIFICANCE - A BREAKTHROUGH IN SCIENCE

Number of Monoclonal Antibodies have been developed in detection and diagnosis of

Parasites like◦ Trichomonas vaginalis ◦ Leishmania donovani◦ Trypanosoma congolense◦ Babesia bovis

Human Viruses like◦ Influenza virus◦ Rotavirus◦ Rabies Virus etc

Animal Viruses like◦ Bovine herpes virus, ◦ Cervine herpes virus type I

MAbs in Pregnancy Testing

MAbs can be used to detect pregnancy with Antibody to Beta HCG.

MAbs in MP Card Test Capture of parasite antigen from

peripheral blood using monoclonal antibodies prepared against a malaria antigen target and conjugated to either a liposome containing selenium dye or gold particles in a mobile phase.

A second or third capture monoclonal antibody applied to a strip of nitrocellulose acts as the immobile phase.

The migration of the antigen-antibody complex in the mobile phase along the strip enables the labeled antigen to be captured by the monoclonal antibody of the immobile phase, thus producing a visible colored line

MAbs in Microalbuminuria Testing

Immunoprecipitation (Micral test): It is based on the colour shift of monoclonal antibody to human albumin labelled with gold.

MAbs IN IMMUNODIAGNOSTIC TESTS

Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.

MAbs in Western Blot

Mab helps to identify a specific molecule/substance in a mixture of substances

MAbs have tremendous application not only in the field of diagnostics but also in ◦ therapeutics and targeted drug delivery systems

for infectious diseases caused by bacteria, viruses, protozoa and for cancer, metabolic and hormonal disorders.

◦ They are also used in the immunological intervention with passive antibody, magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody

THERAPEUTIC SIGNIFICANCE

Auto immune Conditions◦  Rheumatoid arthritis, ◦ Crohn's disease ◦ Ulcerative Colitisby their ability to bind and inhibit TNF Alpha

Acute rejection of organ transplants by inhibiting Interleukin-2 on activated T cells

Allergic Asthma by inhibiting IgE Antibodies Malignancies of solid organs

THERAPEUTIC SIGNIFICANCE…(contd)

Leukemias Lymphomas Osteoporosis Platelet Aggregation Inhibitors

THERAPEUTIC SIGNIFICANCE…(contd)

Anti-cancer monoclonal antibodies can be targeted against malignant cells by several mechanisms:

1)Radioimmunotherapy (RIT) involves the use of radioactively conjugated murine antibodies against cellular antigens to limit radiation exposure.

Murine antibodies were especially chosen, as their high immunogenicity promotes rapid clearance from the body. ◦ Ex:Tositumomab in non-Hodgkins lymphoma.

MAbs in Treatment of Cancer

2)Antibody-directed enzyme prodrug therapy (ADEPT)

It involves the application of cancer associated monoclonal antibodies which are linked to a drug-activating enzyme.

Subsequent systemic administration of a non-toxic agent results in its conversion to a toxic drug, and resulting in a cytotoxic effect which can be targeted at malignant cells

MAbs in Treatment of Cancer

3)Immunoliposomes are antibody-conjugated liposomes.

Liposomes - carry drugs or therapeutic nucleotides - conjugated with monoclonal antibodies - directed against malignant cells.

Tissue-specific gene delivery using immunoliposomes has also been achieved in brain, and breast cancer tissue

MAbs in Treatment of Cancer

DRUGS

Target:◦ VEGF-A(Vascular Endothelial Growth Factor) that

stimulates neovascularisation Indications:

◦ Metastatic Breast,Colon Renal and Brain Cancers◦ Diabetic Retinopathy,Retinopathy of Prematurity

and Choroidal neovascularisation membrane Adv Eff:

◦ Poor wound Healing, Impaired Collateral Formation around Atherosclerotic Vessels, Hypertension and Bleeding

Bevacizumab

Target:◦ TNF-Alpha

Indications:◦ Rheumatoid Arthritis, Ulcerative Colitis, Crohn`s

disease,Psoriatic Arthritis, Ankylosing Spondylitis Adverse Effects:

◦ Infections,Lymphomas,Reactivation of Hep B,Tuberculosis,Drug induced Lupus,Vitiligo

Infliximab

ANTIBODY TARGET USESAbciximab CD41(integrin alpha

receptor)Platelet aggregation inhibitor

Adalimumab TNF ALPHA RA, Crohn's, Psoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, Juvenile Idiopathic Arthritis, Hemolytic disease of the newborn

Alemtuzumab CD 52 CLL

FDA Approved MAbs of Therapeutic Significance

ANTIBODY TARGET USESBasiliximab CD 25(alpha chain of

interleukin 2)Prevention of Organ Transplant Rejection

Belimumab BAFF NHLBevacizumab VEGF -A Metastatic Cancer,

Diabetic RetinopathyBrentuximab Vedotin CD 30 Hematologic CancersCanakinumab IL-1 RACetuximab EGFR Metastatic Colorectal

Cancer and H&N Cancer

Certolizumab pegol TNF alpha Crohns diseaseDaclizumab CD 25(alpha chain of

interleukin 2)Prevention of Organ Transplant Rejection

ANTIBODY TARGET USESDenosumab RANKL Osteoporosis & Bone

CancerEculizumab C5 PNHEfalizumab LFA-1 (CD11a) PsoriasisGemtuzumab CD33 AML

Infliximab TNF Alpha rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, Crohn's disease, ulcerative colitis

Muromonab CD33 Prevention of Organ Transplant Rejection

ANTIBODY TARGET USES

Natalizumab Integrin alpha 4 Multiple Sclerosis,Crohn`s

Omalizumab Ig E Fc region Allergic AsthmaPanitumumab EGFR Colorectal CancerRanibizumab VEGF-A Macular DegenerationRituximab CD20 Lymphomas,LeukemiaTocilizumab IL-6 Receptor RATositumomab CD20 Follicular LymphomaTrastuzumab HER2neu Breast Cancer

RESEARCH SIGNIFICANCE MAbs are used in research purpose also in

analysing ◦ Human Lymphocytes◦ MHC Antigens◦ HLA system

Analysis of antigenic differences between virus and viral related proteins

Antigenic Characterization of viruses, bacteria and parasites

Fever Chills Weakness Headache Nausea Vomiting Diarrhea Rashes Hypo/Hypertension

ADVERSE EFFECTS

DRUG/ TARGET RELATED

SIDE EFFECTS

Immune response against monoclonal Antibodies, producing HAMA ("human anti-mouse antibodies").

Quickly eliminated Immune complexes - Renal Damage Monoclonal antibodies raised in humans

would lessen the problem, but few people would want to be immunized in an attempt to make them, and most of the attempts that have been made have been unsuccessful.

PROBLEMS WITH MONOCLONAL ANTIBODIES

Genetic engineering mouse-human hybrid antibodies to reduce the problem of HAMA.

Chimeric antibodies. ◦ Antigen-binding parts (variable regions) of the mouse

antibody with the◦ Effector parts (constant regions) of a human antibody. ◦ Ex: Infliximab, rituximab, and abciximab 

Humanized antibodies. ◦ Only the amino acids responsible for making the antigen

binding site (the hypervariable regions) of a mouse (or rat) antibody with

◦ the rest of a human antibody molecule. ◦ Ex:Daclizumab, Gemtuzumab, Transtuzumab

HAMA Solved

Letter Meaning

-a- Rat

-e- Hamster

-i- Primate

-o- Mouse

-u- Human

-xi- Chimeric

-zu- Humanized

-axo- Rat/mouse hybrid

--

Source Substem

Fully Human Monoclonal Antibody

Parts of Human Antibody

Parts of Mouse Antibody

Transgenic mice. Have human antibody gene loci inserted into their

bodies (using the embryonic stem cell method). have had their own genes for making antibodies

"knocked out". The result is a mouse that

◦ can be immunized with the desired antigen◦ produces human, not mouse, antibodies against the

antigen◦ can yield cells to be fused with myeloma cells to

manufacture all-human monoclonal antibodies. Phage display is another technique for making all-

human monoclonal antibodies

THE LATEST UPDATE ON MONOCLONAL

ANTIBODIES

The production of recombinant monoclonal antibodies involves technologies, referred to as cloning or phage display/yeast display.

Involves the use of viruses or yeast to create antibodies, rather than mice.

Rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies from which antibodies with desired specificities can be selected.

These techniques can be used to enhance ◦ The specificity with which antibodies recognize antigens ◦ The stability in various environmental conditions ◦ Their therapeutic efficacy and ◦ Their detectability in diagnostic applications 

RECOMBINANT MONOCLONAL ANTIBODIES

NEWER TYPES

mab: whole monoclonal antibody Fab: fragment, antigen-binding (one arm) F(ab')2: fragment, antigen-binding, including

hinge region (both arms) Fab': fragment, antigen-binding, including hinge

region (one arm)

Variable fragments: scFv: single-chain variable fragment di-scFv: dimeric single-chain variable fragment sdAb: single-domain antibody

Bispecific monoclonal antibodies: 3funct: trifunctional antibody BiTE: bi-specific T-cell engager

Antibodies can bind to epitopes expressed at the surface of target cells (as well as to soluble molecules) but are not effective against the peptide fragments that antigen-presenting cells contain tucked within their histocompatibility molecules.

T-cell receptors are the ligands needed for that job So monoclonal antibodies are not effective

against intracellular antigens, e.g. virus-encoded proteins and tumor-specific antigens.

But now progress is being made toward the development of monoclonal T-cell receptors (αβ TCRs).

MONOCLONAL `T` CELL RECEPTORS

Preparing a fusion protein of◦ the engineered TCR conjugated to◦ an effector molecule◦ Cytotoxic Agentto destroy cells expressing the target MHC-peptide complex.

These could then be introduced into a cancer patient to target the tumor-specific antigens or into an AIDS patient to target HIV-infected cells.

Monoclonal TCR

Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially.

The current “big 5” therapeutic antibodies on the market are 

◦BEVACIZUMAB◦TRASTUZUMAB◦ADALIMUMAB◦ INFLIXIMAB◦RITUXIMAB accounted for 80% of revenues in 2006.

In 2007, eight of the 20 best-selling biotechnology drugs in the U.S. are therapeutic monoclonal antibodies.

This rapid growth in demand for monoclonal antibody production has been well accommodated by the industrialization of mAb manufacturing

ECONOMICS

The monoclonal antibody production technology has revolutionized the world of Biotechnology.

Advances in genetic engineering over the years have provided numerous ways to design MAbs that are more robust and efficacious compared with their original murine version.

MAbs have not only been used as diagnostics, therapeutics, research reagents, drug targettor for various infectious diseases but also cancerous, metabolic and hormonal disorders.

MAb technology in conjunction with recombinant DNA technology has successfully  led to the reconstruction of chimeric, humanized and fully human antibodies and has enormous potentials for therapeutic uses.

CONCLUSION

Ophthalmology Dept◦Bevacizumab 1.25mg/0.1ml

Intravitreal injection once monthly for 3 months

Cost Rs.28,000 Radiotherapy Dept

◦Rituximab(100mg/vial) in Non Hodgkin`s Lymphoma

◦ Dosage:375-500 mg/m*2 on day 1of R-CHOP Regimen

◦ Cost Rs.40,000

MAbs used in MBGH

VIDEO 2

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