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Seminar: Topic: Monoclonal
Seminar: Topic: Monoclonal
MONOCLONAL ANTIBODIES
SRINIVASARAO COLLEGE OF PHARMACY
OUTLINE
1. 2. 3.
1. 2. 1. 2.
CONCEPT OF MONOCLONAL ANTIBODIES Structure of immunoglobulin Types Polyclonal Vs monoclonal antibodies DISCOVERY PRODUCTION AND PURIFICATION TECHNICHES Hybridoma technology Purification by chromatographic techniques TYPES 1. Murine 2.Chimeric 3.Humanised 4.Human NOMENCLATURE APPLICATIONS Diagnostic Therapeutic : Rhematologic , hematological ,oncologic INDIVIDUAL MAb s
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ystem to identify and neutralize foreign objects like bacteria and viruses.
Structure of antibody
chain,
The heavy (~50kDa) The light chain (~25kDa).
lambda ().
1. Innate Immunity
2. Acquired Immunity
Primary Response
Secondary Response
immune response can occur when a pathogen is recognized by receptors on B cells. Antigen-derived peptides are then displayed on the B cell surface bound to class II molecules. T helper (Th) cells, have their own receptors that recognize the peptide bound to the B cell class II molecule.
recognizes the antigen, it becomes activated and releases stimulatory molecules called Type II cytokines. activate the B cell to produce and secrete antibodies, which can protect against infection by the same pathogen in the future. Some B cells become memory cells
heterodimeric molecules roximately 150 kDa and are composed of two different kinds of polypeptide chain, called the heavy (~50kDa) and the light chain (~25kDa).
Monoclonal &Polyclonal
Monoclonal antibodies are antibodies that are identical
because they were produced by one type of immune cell (B cell), all clones of a single parent cell
Polyclonal antibodies represent the antibodies from multiple
clones of B lymphocytes, and therefore bind to a number of different epitopes Ex: IV Immunoglobulin
ANTIBODIES
POLYCLONAL. MONOCLONAL.
Derived from different B Lymphocytes cell lines Batch to Batch variation affecting Ab reactivity & titre NOT Powerful tools for clinical diagnostic tests
Classes of Igs :
Classes of Igs IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%)
and IgG4 (4%) , blood and tissue liquid IgA: IgA1 (90%) and IgA2 (10%), external secretions (stomach, intestines, saliva, tears, etc.)IgM: 5-10% of total serum Ig [1.5mg/ml serum conc.] IgD: 1% of proteins in the plasma membranes of Blymphocytes, function unknown [30g/ml serum conc.] 0.2% of total serum Ig. IgE: 0.3g/ml on the surface of plasma membrane of mast cells, play a role in immediate hypersensitivity and defence for parasite
Discovery
The idea of a "magic bullet"
was first proposed by Paul Ehrlich who at the beginning of the 20th century postulated that if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity.
and Niels Kaj Jerne in 1975 who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery hybridoma technology
In 1988 Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients.
Hybridoma technology
Principle
MYELOMA CELLS HAVE LOST
the ability to synthesize hypoxanthine-guaninephosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids Which enables cells to synthesize purines by the salvage pathway here using an extracellular source of hypoxanthine as a precursor
Titre High
BOOST 2 weeks (Pure antigen)
+ 8 - Azaguanine
Immortal Tumor Of Lymphocytes
Myeloma Cells
Myeloma Cells
HGPRTHigh Viability & Rapid Growth
Contd
Purification techniques
Cells, cell debris, lipids,
and clotted material are first removed, typically by filtration with a 0.45 um filter.
Chromatography
Most of the charged
impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography.
contd
A much quicker method of
separation is Protein A affinity chromatography. The antibody selectively binds to Protein A, so a high level of purity is obtained.
However, this method is not
Murine antibody
1. 2. 3. 4.
antibodies Composed of murine Variable regions fused onto human constant regions developed by Recombinant DNA technology Antibodies are Approximately 65% Human origin .
This reduces immunogenicity thus increases
Humanised Mab
Humanised antibodies are
produced by grafting murine hypervariable amino acid domains into human antibodies. of approximately 95% human origin antigens
transferring human immunoglobulin genes into the murine genome, after which the transgenic mouse is vaccinated against the desired antigen, leading to the production of monoclonal antibodies
Nomenclature
naming scheme for assigning generic, or nonproprietary, names to monoclonal antibodies. This naming scheme is used for both the World Health OrganizationsInternational Nonproprietary Names (INN)[5] and the United States Adopted Names (USAN)[1] for pharmaceuticals.
mab.
antibody nomenclature uses different preceding word parts (morphemes) depending on structure and function. These are officially called substems and sometimes erroneously infix
Source substem meaning -a-erat hamster primate mouse human chimeric (human/f oreign)
Stem
circulator -iy system fungus interleuk in inflamm atory lesi ons -o-u-xi-zu-
-mab
immune system
humaniz ed
mul-ne(u)(r)-os-toxa-co(l)-go(t)-go(v)-
-n(e)-* -s(o)-tox(a)-
musculoskel etal system nervous system bone toxin colonic tumor testicular tumor ovarian tumor
-xizu-*
-axo-
-t(u)-
-ma(r)-me(l)-pr(o)-
mammary tumor
melanoma prostate tumor
CAovarian 125 (imitat cancer ion) platelet CD41 (inte aggregatio grin n alpha-IIb) inhibitor TNF-
Abciximab ReoPro
Fab
chimeric
Adalimum Humira ab
mab
human
METHOD OF PREPERATION :
Hybridoma technology :
Principle : Principle MYELOMA CELLS HAVE LOST the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids . Which enables cells to synthesize purines by the salvage pathway here using an extracellular source of hypoxanthine as a precursor
lack HGPRT.
Unfused normal spleen cells cannot grow
Mouse Donor For Generation Of Hybridoma cells HYBRIDOMA TECHNOLOGY ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre
Step 2: -
HYBRIDOMA TECHNOLOGY After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery) Titre too low BOOST(Pure antigen) Titre High BOOST(Pure antigen) 2 weeks PRODUCTION OF MONOCLONAL ANTIBODYreached in Serum
TECHNOLOGY Immortal Tumor Of Lymphocytes + 8 - Azaguanine Myeloma Cells High Viability & Rapid Growth HGPRT- Myeloma Cells PRODUCTION OF MONOCLONAL ANTIBODY
Spleen Cells & Selection of Hybridoma Cells HYBRIDOMA TECHNOLOGY FUSION PEG MYELOMA CELLS SPLEEN CELLS HYBRIDOMA CELLS ELISA PLATE Feeder Cells Growth Medium HAT Medium Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas PRODUCTION OF MONOCLONAL ANTIBODY
Dilution or Expansion HYBRIDOMA TECHNOLOGY A. Clone Each +ve Culture B. Test Each Supernatant for Antibodies C. Expand +ve Clones Mouse Ascites Method Tissue Culture Method PRODUCTION OF MONOCLONAL ANTIBODY
Purification techniques :
Purification techniques Cells, cell debris, lipids, and clotted material are first removed, typically by filtration with a 0.45 um filter.
Chromatography :
Chromatography Most of the charged impurities
are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography.
affinity chromatography.
The antibody selectively binds to Protein A, so a high
Clinical Applications
Future Applications
WESTERN BLOT
IMMUNOFLOURESENCE
IMMUNOFLOURESENCE :
Side effects
more common side effects Allergic reactions, such as hives or itching
Flu-like symptoms, including chills, fatigue, fever, and
Diarrhea
Skin rashes
contd
Rare ---- more serious side effects Infusion reactions. Severe allergy-like reactions can occur and, in very few cases, lead to death
Dangerously low blood cell counts. Decreased red blood cells, white blood cells and platelets
Cardiac complications Certain monoclonal antibodies may cause heart failure and a small risk of MI Bleeding. Some of the monoclonal antibody drugs are designed to stop cancer from forming new blood vessels. There have been reports that these medications can cause bleeding
OKT3
Prevents acute rejection
of kidney transplants
Prevents autoimmune
the Tac subunit of the human high-affinity interleukin-2 (IL-2) receptor It functions as an IL-2 receptor antagonist inhibiting IL-2mediated stimulation of lymphocytes, a critical event in the process of allograft rejection.
It is indicated for the
Rheumatoid arthritis
Psoriasis Ankylosing spondylitis
Crohns disease
IgE in the circulation and prevent them from binding to mast cells and from further degranulation
Palivizumab( synagis)
It is a humanized monoclonal antibody (IgG1) produced by
recombinant DNA technology, directed to an epitope in the A antigenic site of the F protein of respiratory syncytial virus (RSV) Synagis is a composite of human (95%) and murine (5%) antibody sequences
It works by preventing the growth of RSV
Immunomodulation
ADCC CDCC
A.
B.
mAbs can modified for delivery of a toxin, radioisotope, cytokine or other active conjugates.
it is also possible to design bispecific antibodies that
Cetuximab (Erbitux) :
It is a recombinant, human/mouse chimeric
monoclonal antibody that binds specifically to the extracellular domain of the human epidermal growth factor receptor (EGFR)
RITUXIMAB:ANTI CD-20 :
monoclonal antibody that was the first MAb to be approved by the FDA for use in human malignancy . This antibody can sensitize chemotherapyresistant cell lines. Acts by causing B cell apoptosis Used in B cell lymphomas Immune thrombocytopenia Rheumatoid arthritis
trastuzumab
RhuMAb HER-2,93 also known as trastuzumab
(Herceptin), is a humanized antibody derived from a murine monoclonal antibody that recognizes HER-2/neu HER-2/neu (c-erbB-2) , a member of the EGFR family, has been targeted for antibody therapy Myocardial dysfunction --- trastuzumab is not recommended in combination with anthracyclines
A monoclonal antibody Bevacizumab binds VEGF and prevents the interact ion of VEGF to its receptors (Flt 1 and KDR) on the surface of endothelial cells. Approved for use in combination therapy with fluorouracil-based regimens for metastatic ca.colon Other uses Nonsmall-cell lung cancer. Side effects hypertension, grade 1 or 2 proteinur ia, slight increase in bleeding, and impaired surgical wound healing.
ADCC:
ADCC Immunoglobulin's clustered on the surface of
the targeted cells and exposes its tail {F c} region, to be recognized by the Fc receptors present on the surface of the macrophages and neutrophils.
This causes lysis of tumor cell.
RADIOIMMUNOTHERAPY
Involves the use of
non-Hodgkins lymphoma.
ADEPT (Antibody Directed Enzyme Prodrug Therapy) Involves the application of cancer associated monoclonal antibodies which are linked to a drugactivating enzyme. Subsequent systemic administration of a nontoxic agent results in its conversion to a toxic drug, and resulting in a cytotoxic effect which can be targeted at malignant cells.
therapy): ADEPT (Antibody mediated Enzyme prodrug therapy) . An antibody developed against a tumor antigen is linked to a drug -activating enzyme and injected to the blood subsequent systemic administration of non-toxic agent or prodrug results in its conversion to a toxic drug & results in cytotoxic effect
IMMUNOLIPOSOMES
Immunoliposomes are
antibody-conjugated liposomes.
Liposomes can carry
IMMUNOTOXINS:
Immunotoxins are proteins that
contain a toxin along with an antibody that binds specifically to target cells. All protein toxins are work by enzymatically inhibiting protein synthesis. .
antibodies (mAbs) attached to biologically active drugs by chemical linkers with liable bonds. Reduces side effects and show wide therapeutic window Doxorubicin, Duanomycin, Chlorambucil etc. can be conjugated with monoclonal antibodies
attached to a single targeting antibody. But as the number of drug molecules attached per antibody molecule increases, the target binding activity of the antibody decreases. To overcome this problem, carriers like dextran, hydroxymethylpropylamineacrylamine (HPMA), and serum albumin can be used to attach more number of drug molecules per targeting antibody. Antibody with 2-3 drug molecules shown satisfactory tumour binding site
blood .
poor vascular permeability. low and heterogeneous tumor uptake.
administering high doses. Use of certain agents to increase tumor vascular permeability . Tumor retention of antibody conjugates may be improved by inhibition of metabolism, by using more stable linkages. Alternatively, normal tissue retention may be decreases through the use of metabolisable chemical linkages inserted between the antibody and conjugated moiety.
RITUXIMAB:ANTI CD-20
Rituximab (Rituxan) is a humanized anti-CD20 monoclonal antibody that was the first MAb to be approved by the FDA for use in human malignancy
This antibody can sensitize chemotherapy-resistant cell lines. Acts by causing B cell apoptosis
Alemtuzumab (Campath-1H)
Alemtuzumab targets the CD52 glycopeptides, which is highly expressed on T and B lymphocytes. It has been tested as a therapeutic agent for chronic lymphocytic leukemia and promyelocytic leukemias, as well as other non-Hodgkin's lymphomas.
gemtuzumab ozogamicin (Mylotarg)t reatment of acute myeloid leukemia. Approved for the
Gemtuzumab ozogamicin is a humanized, calicheamicin
conjugate that focuses the DNA-damaging act ivity to CD33+ cells. CD33 is an antigen expressed on the surface of 85% to 90% of leukemia progenitor cells, but not on mature granulocytes or non hematopoiet ic cells.
trastuzumab
RhuMAb HER-2,93 also known as trastuzumab
(Herceptin), is a humanized antibody derived from a murine monoclonal antibody that recognizes HER-2/neu HER-2/neu (c-erbB-2) , a member of the EGFR family, has been targeted for antibody therapy Myocardial dysfunction --- trastuzumab is not recommended in combination with anthracyclines
Bevacizumab is a recombinant humanized anti-VEGF- A monoclonal Bevacizumab binds VEGF and prevents the interact ion of VEGF to its
bevacizumab antibody
receptors (Flt -1 and KDR) on the surface of endothelial cells. Approved for use in combination therapy with fluorouracil-based regimens for metastatic ca.colon Other uses Nonsmall-cell lung cancer. Side effects hypertension, grade 1 or 2 proteinur ia, slight increase in bleeding, and impaired surgical wound healing. Potentially life-threatening events (arterial thrombotic events, gastro intestinal perforation, hemoptysis) have occurred in a small number of patients
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