Monoclonal Antibodies

GROUP: 09

PRESENTED TO: MA’AM HUMA

DILSHAD

INTRODUCTION & DISCOVERY

IMMUNE SYSTEM!!!Latin term "IMMUNIS" means EXEMPT, referring to protection against foreign

agents.An integrated body system of organs,

tissues, cells & cell products that differentiates self from non-self & neutralizes potentially pathogenic

organisms.

CELLS OF IMMUNE SYSTEM

WHAT ARE ANTIBODIES OR IMMUNOGLOBULIN ?

Immunoglobulin (Ig) are

structurally related

glycoproteins that function as antibodies.

An antibody is a protein used

by the immune

system to identify & neutralize

foreign objects like bacteria &

viruses.

ANTIBODIES...

Monoclonal antibodies (mAb) are identical because they were

produced by one type of immune cell, all clones of a single

parent cell.

Polyclonal antibodies are derived from

different cell lines. They differ in amino acid

sequence.

DIFFERENCE!!!Monoclonal

Derived from a single B cell clone.

mAb offer reproducible & potentially inexhaustible supply of Ab with exquisite specificity.

Enable development of secure immunoassay systems.

Polyclonal

Derived from different B lymphocytes cell lines.

Batch to batch variation affecting Ab reactivity & titre.

NOT powerful tools for clinical diagnostic tests.

DISCOVERY!!!

The idea of a "magic bullet" was first proposed by Paul

Ehrlich at beginning of 20th century.

He 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 agent of selectivity.

DISCOVERY!!!

George Kohler & Cesar Milstein in 1975 shared the Nobel Prize in Physiology or Medicine in 1984

for discovery of hybridoma technology.

The structure of antibodies

Definitions

Fab = Fragment, antigen binding

Fc = Fragment, crystalline

The Fc fragment specifies other biological activities of the molecule. For example, the Fc fragment may determine whether the antibody simply prevents signaling through a receptor, or alternatively, causes the cell’s destruction through complement fixation or targeting immune effector cells.

Deliver chemotherapy to cancer cells. Ado-trastuzumab emtansine (Kadcyla) is one such drug approved to treat HER2-positive breast cancer. Ado-trastuzumab emtansine contains an

antibody that attaches to the HER2 receptors on the breast cancer cells. The cancer cells then ingest the antibody, which releases a few molecules of chemotherapy.

Deliver radiation to cancer cells. Ibritumomab (Zevalin), approved for non-Hodgkin's lymphoma, combines a monoclonal antibody with radioactive particles. The ibritumomab

monoclonal antibody attaches to receptors on cancerous blood cells and delivers the radiation.

Stop new blood vessels from forming. bevacizumab (Avastin) targets a growth signal called vascular endothelial growth factor (VEGF) that cancer cells send out to attract new

blood vessels. Bevacizumab intercepts a tumor's VEGF signals and stops them from connecting with their targets.

Block growth signals. Cetuximab (Erbitux), a monoclonal antibody approved to treat colon cancer and head and neck cancers, attaches to receptors on cancer cells that accept a certain

growth signal (epidermal growth factor). Blocking this signal from reaching its target on the cancer cells may slow or stop the cancer from growing.

Make the cancer cell more visible to the immune system. rituximab (Rituxan) attaches to a specific protein (CD20) found only on B cells, one type of white blood cell. Certain types

of lymphomas arise from these same B cells. When rituximab attaches to this protein on the B cells, it makes the cells more visible to the immune system, which can then attack .

HOW MONOCLONAL ANTIBODY WORKS??

THE TYPES OF MONO-CLONAL ANTIBODIES

Throughout the

progression of

monoclonal drug

development there have been four

major antibody

types developed:

First generation

murine

Second generation

Chimeric

Humanised

human.

1- According To Evolution

2- According to design

Naked Monoclonal Antibody

Conjugated Monoclonal Antibody

Immune-Toxin Monoclonal Antibody

Murine

monoclonal

antibodi

es (suffix -oma

b)

• Murine derived from mice, these proteins are purified after immunization with antigens Major problems associated with murine antibodies included reduced stimulation of cytotoxicity and the formation complexes after repeated administration, which resulted in mild allergic reactions and sometimes anaphylactic shock.

Chimer

ic monoclonal

antibodi

es (suffixes -

ximab, 

-zumab respectively

)

• Chimeric antibodies are composed of murine variable regions fused onto human constant regions. Human gene sequences, taken from the kappa light chain and the IgG1 heavy chain, results in antibodies that are approximately 65% human. This reduces immunogenicity, and thus increases serum half-life.

Humanize

d m

onoclonal

antibodies (s

uffixes -zum

ab)

• Humanised antibodies are produced by grafting murine hypervariable regions on amino acid domains into human antibodies. This results in a molecule of approximately 95% human origin.

Human m

onoclonal

antibodies (s

uffix 

-umab)

• 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

‘Naked’ Monoclonal Antibodies

‘Naked’ means these antibodies are not fused to a toxin. These are without any drug or radio-active

material attached to them

Naked Monoclonal antibodies can kill cells via a variety of mechanisms,

including: Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), and

direct induction of apoptosis.

Conjugated/ labelled/ loaded Monoclonal Antibodies

Conjugated monoclonal antibodies are those joined to a chemotherapy drug, radioactive particle, or cancer cell killing agent.

Gemtuzumab ozogamicin (Mylotarg)

This monoclonal antibody is

conjugated to the cytotoxic agent calicheamycin

It is used to treat acute myelogenous

leukemia (AML), which is a cancer

of the myeloid line of blood cells.

This monoclonal antibody attacks the

CD33 receptor, which is found in

most leukemic blast cells, but not in

normal hematopoietic stem

cells

Once bound to CD33, the antibody-

calicheamycin complex is

transported inside of the AML cells by

lysosomes.

To facilitate selective release inside of the

cancer cells, calicheamycin is

connected to gemtuzumab by a

chemical linker that is stable at

physiologic pH but is hydrolyzed in the acidic pH of the lysosomes that transport the

antibody-calicheamycin

complex into the cell.

Immune-Toxin Monoclonal Antibody

• 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

Conjugate with pro-drug or enzyme

• An Mab against the CD25 antigen on B cells (and lymphomas)

• Conjugated to either the radio-active iasotope indium-111 or yittrium-90 for treatment of lymphoma patients.

Radio-immune Antibody e.g. ibritumomab

PRINCIPLE

Hybridoma Technology Creates Monoclonal antibodieS

Monoclonal antibodies are typically made by fusing myeloma cell with the spleen cells from a

mouse that has been immunized with desire antigen. However recent advances have

allowed the use of rabbit B cells.

Hybridoma technology is used to produce a hybrid cell. These hybrid cells are produced by fusing B-lymphocyte with tumour cell and they are called as myeloma cells. Thus these hybrid cells have got the ability to produce antibodies due to the B-lymphocyte genetic material and also capacity to divide indefinitely in the culture due to the presence of tumour cell or myeloma cells involved in the production of hybrid cells. Therefore, these hybrid cells produced from hybridoma technology are cultured in laboratory or passaged or subcultured using mouse peritoneal cavity and these cells produces monoclonal antibodies, and this technology is called as hybridoma technology.

Inject the protein into the mouse

Remove the spleen

Identify which spleen cells are producing

antibodiesSeparates these cells

and grow in tissue culture tubes

containing HAT mediumScreen each antibody

for cross reactivity

Select antibody which does not cross react

with any other protien

Principles in Hybridoma technology

Cont’d

Isolation Of B Cells and myeloma cells

• Mice 2 weeks old are immunized with the antigen against with monoclonal antibodies are to be raised by subcutaneous injection. Later B Cells are isolated from the spleen of an imunized mouse. Myeloma cell are isolated from bone marrow. 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. Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium.1 week before cell fusion, myeloma cells are grown in 8-azaguanine. Cells must have high viability and rapid growth. The HAT medium allows only the fused cells to survive in culture.

Selection of hybride cell in HAT meduium

( hypoxanthine aminopterine

thymidine)

• HAT medium is used for selection of hybride cell. Nucleotide synthesis is essential for cell survival .in HAT medium ,aminopterine block the synthesis of purine and pyrimidine from simple sugsar (denovo pathway). But can thrive by utilizing hypoxanthine and thymidine present in the medium by salvage pathway using the enzyme hypoxanthine guanine phosphoribosyl transferace (HGPRT)

How HAT medium work in selection of hybride cell.

Here myeloma cell

are HGPRT deficient , so these cell can not survive in HAT medium

as aminopterine block denovo

pathway

B cells are HGPRT + and can

survive in HAT

medium.after some cell division, B

cell undergo

normal cell death.

Hybride cell has HGPRT

enzymes from B

cells. So only

hybride cell can survive

in HAT medium.

Unfused normal spleen cells

cannot grow

indefinitely because of

their limited life

span

Unfused myeloma

cells cannot grow

because they lack HGPRT.

HYBRIDOMA TECHNOLOGY

• Process by which bulk quantities of targeted antibodies against a specific antigen are produced.Monoclonal antibodies are produced via multiple/identical copies of a certain cell called a hybridoma.

HGPRT=Hypoxanthineguanine

phosphoribosyltransferase

PRODUCTION OF MONOCLONAL ANTIBODY INVOLVE’S:

Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma

cells

ANTIGEN ( Intact cell/ Whole cell

membrane/ micro-organisms ) +

ADJUVANT (emulsification)

The first step in making a hybridoma is to generate antibody producing B cells.This is done by immunizing a mouse against the antigen of interest.Intraperitoneal(IP)injections are the most common method for delivering the antigens in to mice.

Step 2: - Screening Of Mice For Antibody Production

After several weeks of

immunization

Screening Of Mice For Antibody Production

It must be determined if the

mouse is producing antibodies of

interest.Test bleeds are performed and examined for the

presence of antibodies.

If the host is producing the desired antibody,the spleen is removed and dissociated in culture

medium to release the resident B cells.The culture medium also includes cells from a

special mouse myeloma cell line.These tumor

cells can divide indefinitely,but do not

produce antibodies

Immortal Tumor Of Lymphocytes

+ 8 - Azaguanine

Myeloma Cells

High Viability & Rapid Growth

HGPRT

Myeloma Cells

Step 3: - Preparation of Myeloma Cells

Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells &

Selection of Hybridoma Cells

FUSION

PEG

MYELOMA CELLSSPLEEN CELLS

HYBRIDOMA CELLSELISA PLATE

Feeder CellsGrowth Medium

HAT Medium

1. Plating of Cells in HAT selective Medium

2. Scanning of Viable Hybridomas

The next step is to separate the fused

hybridoma cells from the unfused B cells and

myeloma cells.Unfused B cells will die because they lack the ability to survive

in culture.

STEP 5:- SEPARATION OF FUSED HYBRIDOMA CELL AND SCREENING

Surviving hybridomas are separated and individually cultured—one cell per well.These cells are described as clonal

cultures because all of the cells from each well are derived from a single cell,and are therefore identical,or

clonal.

After culturing for a few weeks,when the growing cells can be seen,the culture fluid can

be screened for the presence of desirable antibodies.Screening is the most labor-intensive step in the production of monoclonal antibodies

since fusion can result in thousands of individual hybridoma colonies.Note that

different antibodies may react with different epitopes on the same antigen.

SCREENING

Step 6: - 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

Finally,clones that produce the desired antibody are grown in mass culture

and/or frozen for storage.

DIAGNOSTIC APPLICATION OF MONOCLONAL ANTIBODIES

Monoclonal antibodies are used widely in the diagnostic laboratory.

Monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal and Chlamydia infections.

They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells.

Diagnosis of HIV Infection

HIV antigen is attached to the plate.

Patients serum passed over the plate. Any HIV antibody in the patients serum will attached to the antigen already on the plate.

A second antibody which is specific to the HIV antibody is passed over the plate. This antibody will attach to the concentrated HIV antibody on the plate. This second antibody has an enzyme attached to its structure.

Chromagen dye is passed over the complex of concentrated HIV antibody/conjugated antibody.

The enzyme will turn the chromagen to a more intense colour. The more intense the colour, the greater the HIV antibody level. This would be the a positive result for a HIV test.

Pregnancy Tests

A breakthrough in Diagnostics a monoclonal antibody can be used to detect pregnancy in only 14 days after conception.

A pregnant woman has the hormone human chorionic gonadotrophin (HCG) in her urine.

Monoclonal antibodies to HCG have been produced. These have been attached to enzymes which can later interact with a dye molecule and produce a colour change.

Pregnancy Tests

In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens.

They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody.

Monoclonal antibodies can also be used to purify a substance with techniques called immuno-precipitation and affinity chromatography• The Western blot test and immuno dot blot

tests detect the protein on a membrane.• . monoclonal antibodies, allow successful

diagnosis and treatment of human neuroectodermal tumors

• MAbs were found extremely useful in the rapid outbreak of East Coast Fever (ECF)

Purification of proteins

Therapeutic Uses

Monoclonal antibodies are protein molecules made in the laboratory from hybridoma cells (stable cell lines derived by fusing antibody producing cells from ‐immunised animals with cells that confer immortality and high yield antibody production) or by recombinant ‐deoxyribonucleic acid (DNA) technology.

They are originally derived from the immune system and have a number of unique properties that have stimulated their use in medicine, including the ability to bind specifically and with high affinity to almost any molecular structure.

Therapeutic Uses

They can be made in various expression systems such as bacteria or mammalian cells and modified for enhanced affinity, immune function or half life in blood. ‐

They are made in a homogeneous and reproducible form that allows comparisons across laboratories for diagnosis as well as therapeutics. Current major therapeutic applications of monoclonal antibodies include cancer, chronic inflammatory disease, and infection and they constitute the largest and fastest growing sector of the biological pharmaceutical industry.

Therapeutic Uses

Monoclonal antibodies are proteins with high specificity towards targets.

Monoclonal antibodies have multiple utilities in therapy as they can recognise specific structures in targets such as bacteria, viruses, cancer cells, etc.

Monoclonal antibodies can be produced in large amounts in homogeneous and reproducible form for diagnostic and therapeutic purposes.

Monoclonal antibodies have a clear regulatory path for their approval as therapeutics.

Therapeutic Uses

Monoclonal antibodies can be derived from B cells from immunised ‐animals, humans with autoimmune diseases and de novo by phage display.

Therapeutic monoclonal antibodies can be engineered to be more human like proteins, to increase their affinity, reduce their ‐immunogenicity and increase their half life in the circulation, and ‐can be conjugated with toxin for better lytic effect.

Monoclonal antibodies can mediate antibody mediated cytotoxicity ‐by linking the target cells to cytotoxic cells through their binding sites and Fc sites.

New Research

Researchers have discovered a unique monoclonal antibody that can effectively reach inside a cancer cell, a key goal for these important anticancer agents, since most proteins that cause cancer or are associated with cancer are buried inside cancer cells. Scientists from Memorial Sloan-Kettering Cancer Center and Eureka Therapeutics have collaborated to create the new human monoclonal antibody, which targets a protein associated with many types of cancer and is of great interest to cancer researchers.

Unlike other human therapeutic monoclonal antibodies, which can target only proteins that remain on the outside of cancer cells, the new monoclonal antibody, called ESK1, targets a protein that resides on the inside of the cell.

The ESK1 monoclonal antibody was engineered to recognize WT1 peptides brought to the surface of cancer cells.

SIDE EFFECTS OF MONOCLONAL ANTIBODIES:

All treatments have side effects. Some side effects depend on the type of cell the MAB is targeting. They may also depend on whether the MAB has a drug or radioactive substance attached to it.Monoclonal antibodies are given intravenously (injected into a vein). The antibodies themselves are proteins, so giving them can sometimes cause something like an allergic reaction.

The most common side effect of all monoclonal antibodies is an allergic reaction to the drug. This reaction is most likely to happen when you first have the treatment. You will have paracetamol and an antihistamine drug before you have the treatment to prevent a reaction. If you have a reaction, your doctor or nurse can usually control it by slowing down or stopping the drip for a while.

CONT;

•* Chills * fever * an itchy rash * feeling sick * breathlessness•* Wheezing•* Headaches•* Flushes and faintness•* Changes in blood pressure•* Nausea•* Diarrhea

An allergic reaction can include these symptoms, though you

may not have all of

them

SERIOUS SIDE EFFECTS

Serious, but rare, side effects of monoclonal antibody therapy may

include:•* Infusion reactions. Severe allergy-like reactions can occur and, in very few cases, lead to death. You may receive medicine to block an allergic reaction before you begin monoclonal antibody treatment. Infusion reactions usually occur while treatment is being administered or soon after, so your health care team will watch you closely for a reaction.•* Dangerously low blood cell counts. Low levels of red blood cells, white blood cells and platelets may lead to serious complications.•* Heart problems. Certain monoclonal antibodies may cause heart problems, including heart failure and a small risk of heart attack.•* Skin problems. Sores and rashes on your skin can lead to serious infections in some cases. Serious sores can also occur on the tissue that lines your cheeks and gums (mucosa).•* 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.

Compared with chemotherapy drugs, naked mabs tend to have fewer serious side effects. But they can still cause problems in some people. Some mabs can have side effects that are related to the antigens they target. For example:•* Bevacizumab (avastin®) is an mab that targets a protein called VEGF that affects tumor blood vessel growth. It can cause side effects such as high blood pressure, bleeding, poor wound healing, blood clots, and kidney damage.•* Cetuximab (erbitux®) is an antibody that targets a cell protein called egfr, which is found on normal skin cells (as well as some types of cancer cells). This drug can cause serious rashes in some people.•Conjugated antibodies can be more powerful than naked mabs, but they can also cause more side effects. The side effects depend on which type of substance they’re attached to.

AdvantagesHomogeniety: monoclonal antibody represents a single antibody molecule that binds to antigen with the same affinity and promote the same effectors function

Specificity: the product of a single hybridoma reacts with the same epitope on antigens

Immunizing agent: need to be characterized and is ultimately not needed in large quantities of antibody

Selection: it is impossible to select for specific epitope specificities and generate antibodies against a wide range of antigenic determinants

Antibody production: unlimited quantities of a single well defined monospecific reagent

Dis- Advantages

Immune rejection

Time and effort commitment is very large

Effector function: antibody is monoclonal, it may not produce the desired biological response

Cross reactions: antibodies sometimes display unexpected cross reactions with un-related antigen

Specificity: monoclonal against conformational epitopes on native proteins may lose reactivity with antigen

Affinity: monoclonal antibodies average affinity is lower than polyclonal antibodies

Research 1

Recent advances in molecular biology have lead to the ability to generate molecules with the recognition properties of antibodies by completely in vitro processes.

In addition to being more humane, molecules such as recombinant antibodies rAb and aptamers have certain advantages over animal-generated antibodies, including the fact that they can be made to recognize a wider variety of targets with selected specificity and affinity.

Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than using mice.

Advances in molecular biology have lead to the ability to synthesize antibodies de novo in vitro – completely without the use of animals.

•(1) Creation of an antibody gene library.•(2) Display of the library on phage or cell surfaces.•(3) Isolation of antibodies against the antigen of interest.•(4) Modification of the isolated antibodies; and (5) Scaled up production of selected antibodies in a cell culture expression system.

General production

methods for the

manufacture of non-animal

recombinant antibodies can

be broken down into five general steps:

Research 2

Classical therapeutic modalities such as surgery, radiation, and chemotherapy not only fail to cure the great majority of malignant tumors, but their employment often leads to severe and debilitating side effects.

The severe cancer related morbidity is also in direct correlation with the use of x-radiation and chemotherapy, making them less than ideal forms of therapy.

The development of hybridoma technology and the advances in monoclonal antibody (MoAB)

production have revitalized the initial concept of Ehrlich concerning

the existence of cancer cell-targeted, specific "magic bullets".

Entirely new approaches to cancer therapy that are neoplastic cell-

directed, and specifically lethal to malignant cells and less toxic to

normal tissues are being observed and developed, adhering to the old

prayer: "Destroy the diseased tissues, preserve the normal."

•Immune reaction directed destruction of cancer cells.•Interference with the growth and differentiation of malignant cells.•Antigen epitope directed transport of anti-cancer agents to malignant cells.•Anti-idiotype vaccines.•Development of engineered (humanized) mouse monoclonals for anti-cancer therapy.

Immunotherapy as a modality of cancer therapy

has already been developed and proven to be

quite effective. Strategies for

the employment of antibodies for

anti-cancer immunotherapy

include:

In addition, a variety of different agents (e.g. toxins, radionuclides, chemotherapeutic drugs) have been conjugated to mouse and human MoABs for selective delivery to cancer cells.

Finally, we propose that MoAB-based immunotherapy be accepted as a conventional form of therapy and employed not only in terminal cancer patients but also, for instance, during and following surgical resection.