Monoclonal antibodies Clinical Significance
-
Upload
drvicky666 -
Category
Education
-
view
3.195 -
download
3
Transcript of Monoclonal antibodies Clinical Significance
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
THANK YOU