Monoclonal AntibodiesMonoclonal Antibodies

In Nuclear Medicine – In Nuclear Medicine – Understanding The BasicsUnderstanding The Basics

Information on You TubeInformation on You Tube

Suggested review prior to this lectureSuggested review prior to this lecture Role of B LymphocytesRole of B Lymphocytes

• http://www.youtube.com/watch?v=Z36dUduOk1Y

Production of Monoclonal Antibodies Production of Monoclonal Antibodies • http://www.youtube.com/watch?v=O-SrP

qJuEVg

Basic ConceptsBasic Concepts

Antibodies [Ab] (Immunoglobulins [Ig]) are Antibodies [Ab] (Immunoglobulins [Ig]) are produced by plasma cells in response to produced by plasma cells in response to foreign substances (Antigens [Ag])foreign substances (Antigens [Ag])

Ag are usually 1,000 daltons or more in sizeAg are usually 1,000 daltons or more in size Ig possess specific binding regions on the Ig possess specific binding regions on the

surface that recognize the shape of particular surface that recognize the shape of particular sites (determinants) on the surface of an Agsites (determinants) on the surface of an Ag

Ab binds to the Ag in an immunological Ab binds to the Ag in an immunological response destroying the Agresponse destroying the Ag

Ab ResponseAb Response

Ag usually have several determinants or Ag usually have several determinants or epitopesepitopes

Each epitope stimulates one or more B Each epitope stimulates one or more B lymphocyteslymphocytes

B lymphocytes can differentiate into B lymphocytes can differentiate into plasma cells that secrete a specific Ig plasma cells that secrete a specific Ig response to a determinate on the Agresponse to a determinate on the Ag

Hence B lymphocytes create plasma Hence B lymphocytes create plasma cells and the plasma cells produce a cells and the plasma cells produce a host of different Ab in response to the host of different Ab in response to the AgAg

How Antibodies are developedHow Antibodies are developed

Using a mouse or rabbit it is immunized with Using a mouse or rabbit it is immunized with Ag agent – ex. Cancerous tissue is placed Ag agent – ex. Cancerous tissue is placed underneath the skinunderneath the skin

This creates an Ab to Ag response within the This creates an Ab to Ag response within the hosthost

Serum can then be extracted from the host Serum can then be extracted from the host that has the Abs which were created from the that has the Abs which were created from the different epitopes on the Ag surfacedifferent epitopes on the Ag surface

This is known as a polyclonal response This is known as a polyclonal response because there are many types of Abs that because there are many types of Abs that were produced from the inoculation of a were produced from the inoculation of a specific Ag specific Ag

Refer to the image on the next side to define Refer to the image on the next side to define the processthe process

Ab To Ag ResponseAb To Ag ResponseThis side shows the mouse responseto the Ag that createspolyclonal Ab. Important points:•Ag is injected into host •Lymphocytes respond to Ag and produce Abs•Taking lymphocytes from host you can fuse this with Myeloma Cells resulting in hybrid Ab

•These polyclonal Abs can be separated into MoAbs

•Refer to the side next tonote MoAbProduction

Where Do MoAbs Come From?Where Do MoAbs Come From? Lymphocytes or Lymphocytes or

Plasma cells are Plasma cells are extracted from the extracted from the mouse and fused mouse and fused with myeloma cellswith myeloma cells

This creates a hybrid This creates a hybrid myeloma cellsmyeloma cells

They are clonedThey are cloned Specific MoAbs are Specific MoAbs are

then grown in then grown in cultureculture

A closer look at the process in which MoAbs are produced.

The process is discussed on the following slides

Creating the MoAb For Medical Creating the MoAb For Medical UseUse

Extract the splenic lymphocytes from an Extract the splenic lymphocytes from an immunized mouse along with the myeloma cell immunized mouse along with the myeloma cell lineline

Fusing these cells creates a hybridoma cellsFusing these cells creates a hybridoma cells Fusing occurs in a polyethylene glycol solution Fusing occurs in a polyethylene glycol solution

where the cell will multiplewhere the cell will multiple Selected hybridoma cells are then grown in a Selected hybridoma cells are then grown in a

hypoxanthene-aminopterin-thymidine (HAT) hypoxanthene-aminopterin-thymidine (HAT) medium (only fused cells survive)medium (only fused cells survive)

These cells can then be separated for assay These cells can then be separated for assay and are re-cultured (re-cloned) until the right and are re-cultured (re-cloned) until the right MoAb is foundMoAb is found

The Immunoglobulins (Ig)The Immunoglobulins (Ig)

There many types of Igs: IgG, IgM, There many types of Igs: IgG, IgM, IgE, IgA, and IgDIgE, IgA, and IgD

Usually hybridomas are developed Usually hybridomas are developed from some form of IgG or its subclassfrom some form of IgG or its subclass

The next slide demonstrates the The next slide demonstrates the structure of an IgG MoAbstructure of an IgG MoAb

IgG StructureIgG Structure

Variable region (light Variable region (light chain) respond to the chain) respond to the different to the different to the epitopes on the Ag epitopes on the Ag surfacesurface

Constant or heavy Constant or heavy region remains the region remains the samesame

These chains are These chains are held together by a held together by a disulfide bondsdisulfide bonds

Note that the IgG Note that the IgG structure can be structure can be fragmented via fragmented via pepsin or papainpepsin or papain

Fragmented: F(ab’)Fragmented: F(ab’)2 2 ,Fab, and ,Fab, and

Fc Fc Removal of most or all of the heavy change Removal of most or all of the heavy change

causescauses• Reduce HAMA responseReduce HAMA response• Allows for faster clearance after injectionAllows for faster clearance after injection

MoAbs have been created from all of the MoAbs have been created from all of the above mentioned typesabove mentioned types

Can you identify a whole IgG and fragmented Can you identify a whole IgG and fragmented IgG used in nuclear medicine?IgG used in nuclear medicine?

Discuss some of its imaging propertiesDiscuss some of its imaging properties Whole IgG is (~50,000 daltons or greater) that Whole IgG is (~50,000 daltons or greater) that

metabolize in the liver, while fragmented are metabolize in the liver, while fragmented are much smaller and quickly excreted by the much smaller and quickly excreted by the kidneyskidneys

Some other pointsSome other points

Total mol.wt. of an IgG is up to ~150,000 Total mol.wt. of an IgG is up to ~150,000 daltonsdaltons

Affinity refers to the strength of attraction Affinity refers to the strength of attraction between the Ab-Agbetween the Ab-Ag

Avidity refers to the integrity of the Ab-Ag Avidity refers to the integrity of the Ab-Ag bondbond

Both affinity and avidity are important in Both affinity and avidity are important in order for specific/strong tag to occur order for specific/strong tag to occur between the Ab-Agbetween the Ab-Ag

Comment on HAMAComment on HAMA A mouse contains murine AbsA mouse contains murine Abs Human response to this could be a Human response to this could be a

human anti-body (HAMA) reactionhuman anti-body (HAMA) reaction Human-human hybridomas should be Human-human hybridomas should be

considered to reduce the HAMA considered to reduce the HAMA responseresponse

HAMA is an allergic reaction HAMA is an allergic reaction • Anaphylactic is the most server and if left Anaphylactic is the most server and if left

unchecked could cause deathunchecked could cause death• Interferes with imaging can also occur Interferes with imaging can also occur

creating a false negative image (the body creating a false negative image (the body has prevented Ab-Ag to occur because has prevented Ab-Ag to occur because HAMA interferes)HAMA interferes)

Finding the Right Radionuclide TagFinding the Right Radionuclide Tag

Consider Consider • Therapy vs. diagnostic (beta vs. gamma)Therapy vs. diagnostic (beta vs. gamma)• Type of radionuclide (Tc99m vs. In111)Type of radionuclide (Tc99m vs. In111)• Tagging whole vs. fragmented IgGTagging whole vs. fragmented IgG

Therapy vs. DiagnosticTherapy vs. Diagnostic

Therapy Therapy • If beta radiation is used particle If beta radiation is used particle

radiation can destroy the diseaseradiation can destroy the disease• Requires a strong/stable Ab-Ag reactionRequires a strong/stable Ab-Ag reaction

DiagnosticDiagnostic• Gamma radiation is usedGamma radiation is used• Disease is identifiedDisease is identified• Requires a strong/stable Ab-Ag reactionRequires a strong/stable Ab-Ag reaction

Type of RadionuclideType of Radionuclide

If the radio-MoAb is the entire IgGIf the radio-MoAb is the entire IgG• Takes a long time to clear or get a good Takes a long time to clear or get a good

target to background (72 or more hours)target to background (72 or more hours)• Requires a radionuclide such as In111Requires a radionuclide such as In111

Fragmented IgGFragmented IgG• Clears quickly and gives a better target Clears quickly and gives a better target

to background (usually within 24 hours)to background (usually within 24 hours)• Tc99m can be used Tc99m can be used

More on the Whole IgG MoAbMore on the Whole IgG MoAb

When using In111When using In111• DTPA is used so that the In111 tags to the DTPA is used so that the In111 tags to the

heavy change of the MoAbheavy change of the MoAb• In111 – DTPA – MoAb (on the heavy change)In111 – DTPA – MoAb (on the heavy change)• In111 is then introduced and tags to the In111 is then introduced and tags to the

DTPADTPA• Remember In111 must be used if the MoAb Remember In111 must be used if the MoAb

being used requires significant filtering by being used requires significant filtering by the body, over time the body, over time

• What other gamma emitters could we use if What other gamma emitters could we use if a whole IgG MoAb is being used?a whole IgG MoAb is being used?

Complications of the Radioactive Complications of the Radioactive TagTag

Radiolabeling may alter the biological activity Radiolabeling may alter the biological activity of the MoAb, rendering it either less infective of the MoAb, rendering it either less infective

Immunoreactive fraction is a concern and Immunoreactive fraction is a concern and results when free MoAb dissociates from the results when free MoAb dissociates from the radioactive tagradioactive tag

Immunospecificity is another issue where Immunospecificity is another issue where specificity of the agent can be lost. This can specificity of the agent can be lost. This can occur by any one of the following occur by any one of the following • Blood flowBlood flow• MetabolismMetabolism• Capillary permeabilityCapillary permeability

Target to Background RatiosTarget to Background Ratios Minimal requirement 2:1, but 5:1 is preferredMinimal requirement 2:1, but 5:1 is preferred Digital subtraction and image contrast can help, Digital subtraction and image contrast can help,

however, a higher false-positive may occurhowever, a higher false-positive may occur Theoretically 100:1 to 1000:1 ratios should be Theoretically 100:1 to 1000:1 ratios should be

attained, however, this has never happenedattained, however, this has never happened To make an idea MoAb consider the following: To make an idea MoAb consider the following:

MoAb clearance, reducing background, reducing MoAb clearance, reducing background, reducing dosimetry, and reducing HAMA favor the dosimetry, and reducing HAMA favor the fragmented MoAbfragmented MoAb

Clinical StatusClinical Status MoAbs have been created to identifyMoAbs have been created to identify

• Lung CancerLung Cancer• Prostate CancerProstate Cancer• Colon CancerColon Cancer• InfectionInfection• LymphomaLymphoma

Still lacks sensitivity and specificityStill lacks sensitivity and specificity Is it the poor man’s PET?Is it the poor man’s PET? When you look at the sensitivity and When you look at the sensitivity and

specificity of PET over MoAbs, you might specificity of PET over MoAbs, you might conclude that MoAbs are the inferior conclude that MoAbs are the inferior scan (see PET lecture)scan (see PET lecture)

Review of the IssuesReview of the Issues Excessive background (more so with whole IgG)Excessive background (more so with whole IgG) Ionization of the radioactive tag (losses Ionization of the radioactive tag (losses

specificity)specificity) Cross-reactivity with non-specific Ag (goes where Cross-reactivity with non-specific Ag (goes where

you don’t want it to)you don’t want it to) Variation of expression Ab-Ag can result in a false Variation of expression Ab-Ag can result in a false

–negative study–negative study HAMA response to the IgG reduces image qualityHAMA response to the IgG reduces image quality Alternate routes of administration should be Alternate routes of administration should be

consideredconsidered

ProstaScint and CEA ScanProstaScint and CEA Scan

This is an example of

Prostate mets

MDP vs MoAbMDP vs MoAb

http://www.nature.com/ncpuro/journal/v3/n4/fig_tab/ncpuro0452_F2.html

For more information you can access the following article:

“Monoclonal Antibodies in Nuclear Medicine”, by AM. Keenan,

Et al. Jour NM, May 1985

http://www.med.harvard.edu/JPNM/physics/pharms/radpharm/antibod/FDAnov96.html

Return to the Table of Content