SEMINAR

TOPIC: MONOCLONAL ANTIBODIES

S.MANOJ KUMAR M PHARMACY 1ST YEAR PHARMACEUTICALTECHONOLOGY SHIFT2 ROLLNO;08

MONOCLONAL ANTIBODIES
SRINIVASARAO COLLEGE OF PHARMACY

NAME:MANOJ KUMAR.S ROLLNO:08 SHIFT-II M PHARMACY 1stYEAR PHRMACEUTICALTECHNOLOGY

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

The Perfect World

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The Real World

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What are antibodies?

antibody is a protein used by the immune sAn

ystem to identify and neutralize foreign objects like bacteria and viruses.

ach antibody recognizes a specific antigen unique to its target.

Structure of antibody

 IgG Ab are large heterodimeric molecules

approximately 150 kDa and

Are composed of two different kinds of polypeptide

chain,
The heavy (~50kDa) The light chain (~25kDa).

There are two types of light chains, kappa () and

lambda ().

THE IMMUNE SYSTEM

The Latin term IMMUNIS means EXEMPT, referring to protection against foreign agents. DEFINITION: - The integrated body system of organs, tissues, cells & cell products that differentiates self from non self & neutralizes potentially pathogenic organisms The Immune System consists of

1. Innate Immunity
2. Acquired Immunity

Primary Response
Secondary Response

 Initiation of the humoral

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.

CD4+ T cells, also called

 When the CD4+ T cell

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

Type II cytokines, in turn,

Structure of immunoglobulin's & types

 appIgG Ab are large

heterodimeric molecules roximately 150 kDa and are composed of two different kinds of polypeptide chain, called the heavy (~50kDa) and the light chain (~25kDa).

There are two types of

light chains, kappa () and lambda ().

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

Derived from a single B cell clone

mAb offer Reproducible, mAb offer Reproducible, Predictable & Potentially Predictable & Potentially inexhaustible supply of Ab inexhaustible supply of Ab with exquisite specificity with exquisite specificity Enable the development of secure immunoassay systems.

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.

 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.

Georges Khler Csar Milstein,

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

 The selective culture medium is

called HAT medium because it contains Hypoxanthine, Aminopterin, and Thymidine

Unfused myeloma cells cannot

grow because they lack HGPRT.

Unfused normal spleen cells

cannot grow indefinitely because of their limited life span.

PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells
ANTIGEN ( Intact cell/ Whole cell membrane/ microorganisms ) + ADJUVANT (emulsification)

Ab titre reached in Serum

PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY Step 2: - Screening Of Mice For Antibody Production
After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery)

Titre too low

BOOST (Pure antigen)

Titre High
BOOST 2 weeks (Pure antigen)

PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY Step 3: - Preparation of Myeloma Cells

+ 8 - Azaguanine
Immortal Tumor Of Lymphocytes

Myeloma Cells

Myeloma Cells
HGPRTHigh Viability & Rapid Growth

PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells
PEG FUSION SPLEEN CELLS MYELOMA CELLS Feeder Cells Growth Medium
1. Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas

HYBRIDOMA CELLS ELISA PLATE 2. HAT Medium

PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY Step 5: - Cloning of Hybridoma Cell Lines by Limiting Dilution or Expansion A. Clone Each +ve Culture B. Test Each Supernatant for Antibodies

C. Expand +ve Clones

Tissue Culture Method Mouse Ascites Method

PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY

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

advisable for antibodies that are easily damaged

Types of Monoclonal Antibodies

Murine antibody

1. 2. 3. 4.

Whole of the antibody is of murine origin

Major problems associated with murine antibodies include reduced stimulation of cytotoxicity Formation of complexes after repeated administration Allergic reactions Anaphylactic shock

 This reduces immunogenicity and

thus increases serum half-life.

Chimeric monoclonal antibody:

Chimeric monoclonal antibody Chimeric

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

serum-half life Ex. Basiliximab, Cetuximab

Humanised Mab
Humanised antibodies are

produced by grafting murine hypervariable amino acid domains into human antibodies. of approximately 95% human origin antigens

This results in a molecule

These bind weakly to the

Human Monoclonal antibody

Human monoclonal antibodies are produced by

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

nomenclature of monoclonal antibodies

The nomenclature of monoclonal antibodies is a

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.

 In All monoclonal antibody names end with the stem -

mab.

Unlike most other pharmaceuticals, monoclonal

antibody nomenclature uses different preceding word parts (morphemes) depending on structure and function. These are officially called substems and sometimes erroneously infix

Prefix old -anibi-ba(c)-ci(r)variable -fung-ki(n)-les-li(m)-

Target substem new -b(a)-c(i)-f(u)-k(i) -l(i)meaning

Source substem meaning -a-erat hamster primate mouse human chimeric (human/f oreign)

Stem

angiogen esis (inhi bitor) bacteriu m

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-*

chimeric/hu manized hybrid rat/mouse hybrid (see trifunctio nal antibody)

-axo-

-t(u)-

-ma(r)-me(l)-pr(o)-

mammary tumor
melanoma prostate tumor

includes approved and investigation al drugs

Name 3F8 Abagovom ab[1] Trade name ? mab Type Source mouse mouse Target GD2 Use neuroblast oma

CAovarian 125 (imitat cancer ion) platelet CD41 (inte aggregatio grin n alpha-IIb) inhibitor TNF-

Abciximab ReoPro

Fab

chimeric

Adalimum Humira ab

mab

human

rheumatoi d arthritis etc

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

 The selective culture medium is called HAT

medium because it contains, Hypoxanthine, Aminopterin , and thymidine

Unfused myeloma cells cannot grow because they

lack HGPRT.
Unfused normal spleen cells cannot grow

indefinitely because of their limited life span.

PRODUCTION OF MONOCLONAL ANTIBODY

Step 1: - Immunization Of Mice & Selection Of

Mouse Donor For Generation Of Hybridoma cells HYBRIDOMA TECHNOLOGY ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre

Step 2: -

Screening Of Mice For Antibody Production

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

 Step 3: - Preparation of Myeloma Cells HYBRIDOMA

TECHNOLOGY Immortal Tumor Of Lymphocytes + 8 - Azaguanine Myeloma Cells High Viability & Rapid Growth HGPRT- Myeloma Cells PRODUCTION OF MONOCLONAL ANTIBODY

Step 4: - Fusion of Myeloma Cells with Immune

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

 Step 5: Cloning of Hybridoma Cell Lines by Limiting

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.

 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 advisable for antibodies

that are easily damaged

Applications of Monoclonal Diagnostic Applications Antibodies Biosensors & Microarrays

Therapeutic Applications
Transplant rejection Cardiovascular disease Cancer Infectious Diseases Inflammatory disease

Clinical Applications

Purification of drugs, Imaging the target

Future Applications

Fight against Bioterrorism

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

muscle aches and pains

Nausea

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

destruction of islet cells in type I Diabetes mellitus

DACLIZUMAB: ANTI IL-2

Daclizumab binds specifically to

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

prophylaxis of acute organ rejection in patients receiving renal transplants

ABCIXIMAB IIb-IIIa Anti platelet factor(GP

inhibitor) is the Fab fragment of the
chimeric human-murine monoclonal antibody (Reopro) INDICATIONS:

Adjunct to PCI for the prevention of

cardiac ischemic complications

In patients undergoing PCI In patients with unstable angina not

responding to conventional medical therapy when PCI is planned within 24 hours

INFLIXIMAB: ANTI-TNF AGENT

Rheumatoid arthritis
Psoriasis Ankylosing spondylitis

Crohns disease

OMALIZUMAB: ANTI IgE

Acts by binding to free

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

Monoclonal antibodies for cancer treatment

Mechanisms that could be responsible for the cancer treatment

Binding to a critical receptor and blocking down stream

signaling Down regulation of receptors

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

can bind with their Fab regions both to target

antigen and to a conjugate or effector cell

PRINCIPLES OF CANCER THERAPY

 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 :

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 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

 Bevacizumab is a recombinant humanized anti-VEGF-

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

radioactively conjugated Murine antibodies against cellular antigens

Ex; Tositumomab -----

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.

 ADEPT (Antibody mediated Enzyme prodrug

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

drugs or therapeutic nucleotides and when conjugated with monoclonal antibodies

 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. .

 Any drug conjugates: Antibody drug conjugates are monoclonal

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

 DRUG LOADING: Generally, a higher number of drug molecules are

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

Problems of delivery of monoclonal antibodies:

slow elimination of monoclonal antibodies from the

blood .
poor vascular permeability. low and heterogeneous tumor uptake.

Cross reactivity with normal tissues.

Metabolism of monoclonal antibody conjugates

 Tumor uptake may be increased through

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

Used in B cell lymphomas

Immune thrombocytopenia Rheumatoid arthritis

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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